Class 12 Medical Biology - Biotechnology: Principles And Processes

Answer in 3 to 5 sentences:
Draw a neat labelled diagram of a typical agarose gel electrophoresis.

In agarose gel electrophoresis, agarose is used as a matrix. The sample is added in the slot and current is applied to it. The smaller molecules move faster and the larger molecules are retarded. In this method, separation is based on charge and size of the molecules. DNA, RNA and proteins can be separated using this technique.

Manipulating with nucleic acid is a trend in biotechnology.
(a) Name any one organism used as vector.
(b) What are DNA polymerase ?

a) Bacteriophage can be used as vectors.

b) DNA polymerase is an enzyme involved in DNA replication. It is responsible for forming new copies of DNA from nucleic acids.

How will you transfer a whole chromosome into a fertilized egg?

Certain genes or the part of the chromosomes can be introduced in the cells to show the respective feature. For the transfer of whole chromosomes, metaphase cells are selected. These cells are subjected to hypotonic lysis and individual chromosomes or fragments are isolated. The fragments of the chromosomes are incubated with whole cells/ eggs for transfection. This allows the egg cells to take up the chromosomes from the medium.

Name the bacterium that yields a thermostable DNA polymerase.

The bacterium that yields a thermostable DNA polymerase is Thermus aquaticus

What is main function of gel electrophoresis?

The main function of gel electrophoresis is to separate DNA fragments using electric current according to their size

What is particle gun?

Particle gun is a technique of bombarding micro-particles of gold or tungsten coated with foreign DNA with great velocity into the target cells

What chemical is used in vectorless gene transfer?

Polyethylene glycol helps in transfer of foreign DNA into the host cell.

Which cloning vector was discovered for the first time?

The first widely used Cloning vector $$pBR322$$ that contains around 4300 base pairs. Named after the scientists Boliver and Rodrigues

Name an eukaryotic organism that has plasmids, and can be used as a host in gene cloning experiments.

Yeast is the eukaryotic organisms that has plasmids and can be used as host in gene cloning experiments

What was the first recombinant DNA based product, produced and marketed in India?

The first recombinant DNA based product , produced and marketed in India is Hepatitis B virus vaccine

What is called a 'probe'?

It is a single standard DNA or RNA tagged with a radioactive molecule, which is complementary to the DNA in a clone of cells.

Name the compound used for staining the isolated DNA in the gel.

The compound used for staining isolated DNA in a gel is Ethidium bromide

(a) Why must a cell be made 'competent' in biotechnology experiments? How does calcium ion help in doing so?
(b) State the role of biolistic gun in biotechnology experiments.

a) The cell must be made competent in biotechnology experiments because DNA being a hydrophilic molecule, cannot pass through cell membrane. Hence, the cell should be made competent to accept the DNA molecule. Calcium ions play an important role in neutralizing the natural negative charges possessed by both DNA and cell surface to avoid electrostatic repulsion between the two.

b) The biolistic gun has been developed to introduce rDNA into mainly plant cells by using a gene / particle gun. In this method, microscopic particles of gold / tungsten are coated with the DNA of interest and bombarded onto the cells.

How is the amplification of a gene sample of interest carried out using Polymerase Chain Reaction (PCR)?

Polymerase chain reaction (PCR) is the process in which the amplification of the gene of interest is carried out with two sets of primers and a thermostable DNA polymerase enzyme Taq polymerase. The process involves the following steps:

Denaturation: The double-stranded DNA is heated up to 94 $$^o$$C which causes the hydrogen bonds to break and the two strands get separated.
Annealing: The two sets of primers are added which bind to the appropriate complementary segment of DNA strand at 54$$^0$$C.
Extension: The Taq polymerase enzyme polymerizes the nucleotide chain using the nucleotides provided in the medium and by using the template strand at 72$$^0$$C.

Name the source organism that possesses Taq polymerase. What is so special about the function of this enzyme?

Taq polymerase is a thermostable DNA polymerase which is named after the bacterium from which it was first originally isolated -Thermus aquaticus.

It is used in the polymerase chain reaction or PCR, a method which is used to amplify the quantity of short segments of DNA. This enzyme is able to withstand the protein-denaturing conditions (high temperature) required during PCR or it is thermostable.

How does a restriction nucleases function? Explain.

Restriction endonuclease, is the most widely used enzyme for the process of gene modification. It recognizes a specific sequence of nucleotides which is known as recognition site and produces a double strand nick in the respective DNA. The action of restriction endonuclease can be on palindromic sequences. For example, EcoRI cuts the DNA at the following palindromic sequence:
5' GAATTC 3'

3'CTT AAG 5'

Why is the enzyme cellulase needed for isolating genetic material from plant cells and not from the animal cells?

The plant cells have the cell wall and so to digest it, cellulase is required. Animals do not have the cell wall and so cellulase is not required.

Expand the following and mention one application of each:
(i) PCR (ii) ELISA

PCR - Polymerase Chain Reaction
Application: It is used in various applicatons like detection, sequencing, micro-arrays, paternity testing etc.

ELISA - Enzyme Linked ImmunoSorbent Assay
Application: ELISA is used to determine the serum antibody concentrations in a virus test.

How are 'sticky ends' formed on a DNA strand? Why are they so called?

Sticky ends are produced by the restriction enzymes. The restriction enzymes cut the strand of DNA a little away from the center of the palindromic sequences but between the same two bases on the opposite strands. This leaves single-stranded portions at the ends. There are overhanging stretches called 'sticky ends' on each strand.

These are called sticky ends because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

Explain with the help of a suitable example the naming of a restriction endonuclease.

Restriction endonucleases are named as follows:

$$1^{st}$$ alphabet represents the genus of the organism from which the enzyme is isolated.
$$2^{nd}$$ and $$3^{rd}$$ alphabet represents the species of the organism.

$$4^{th}$$ alphabet represents the strain.

The roman number represents the order of isolation or discovery of the enzyme.

For example, EcoRI is the restriction enzyme, derived from Escherichia coli, strain R and is the first to be isolated.

Mention the type of host cells suitable for the gene guns to introduce an alien DNA.

To introduce an alien DNA, undifferentiated plant cells are the most suitable host cells for the gene guns. This is so because the undifferentiated plant cells have a cell wall which can be easily broken down by bombarding them with high-velocity micro-particles of gold or tungsten coated with DNA in a gene gun. The development of the tertiary cell wall in the mature plant cell is a hindrance to the introduction of foreign DNA.

Name and describe the technique that helps in separating the DNA fragments formed by the use of restriction endonuclease.

The technique used for separating DNA fragments in the lab is gel electrophoresis.

Electrophoresis enables to distinguish DNA fragments of different lengths.
DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size.
The use of dyes or radioactive labels enables the DNA on the gel to be seen after they have been separated. They will appear as bands on the gel.
A DNA marker with fragments of known lengths is usually run through the gel at the same time as the samples. By comparing the bands of the DNA samples with those from the DNA marker, one can work out the approximate length of the DNA fragments in the samples.

State the role of DNA ligase in biotechnology.

DNA ligase is used to join two fragments of DNA to seal the gaps between them. Thus, they are known as molecular glues. Only the matching DNA fragments with complementary ends can be joined with DNA ligase. Ligation is a natural process during replication process where Okazaki fragments are joined together. Ligation is carried out for the purpose of cloning in the laboratory.

Draw a schematic sketch of pBR 322 plasmid and label the following in it.
(a) Any two restriction sites
(b) Ori and rop genes
(c) An antibiotic resistant gene

The given image is a diagram of pBR 322 plasmid showing EcoRI and HindIII restriction sites, Ori and rop genes and tetracycline and ampicillin resistant genes.

Make a chart(with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces.

The name of the restriction enzyme is Bam H1.

The substrate for BamH1 enzyme is GGATCC. BAM H1 enzyme can make cut or nick on the G nitrogen base of restriction site and produces cut with sticky ends.

Do eukaryotic cells have restriction endonucleases? Justify your answer.

Restriction endonucleases are naturally occurring defence mechanism of bacteria to digest any foreign DNA molecule. Restriction endonucleases recognize specific sequences, mostly 4-6 bp, in the DNA and cut it into fragments by breaking the phosphodiester linkage between two successive nucleotides of DNA. As these restriction sites may be present in bacterial DNA itself, DNA methylase enzyme carry out methylation of DNA to protect own DNA in bacteria from restriction digestion. Eukaryotic DNA is highly methylated and therefore, these enzymes are not found in eukaryotes.

Explain briefy.
(a) PCR
(b) Restriction enzymes and DNA
(c) Chitinase

(a) PCR - Polymerase chain reaction technique amplify the DNA from one copy to more than one in three steps namely denaturation of target DNA (thermal cycle to separate the DNA strands), annealing of primers to the ssDNA and polymerization (extension of primer into complete DNA strand complementary to the template strand).

(b) Restriction enzymes recognize specific sequences, mostly 4-6 bp, in the DNA and cut it into fragments by breaking the phosphodiester linkage between two successive nucleotides of DNA. DNA is polymer of deoxyribonucleotides and serves as genetic material

Explain the use of Agrobacterium tumifaciens in gene cloning.

Agrobacterium tumefaciens is a plant pathogen and most effective as plant genetic engineer. It causes the formation of crown gall in plants. The tumor-inducing plasmid Ti can be used as a useful cloning vector in gene cloning in plants. It introduces a piece of DNA which transforms normal plant cells into tumor cells. The Ti plasmid contains genes for the synthesis of auxin and cytokinin hormone and thus its introduction in a plant helps it to produce its own nutrient machinery.

Give a brief account of the tools of recombinant DNA technology.

Recombinant DNA technology is the method which helps in the production of transgenic organisms. There are certain tools which are used in this technique. The basic steps involved in the process of DNA technology are as follows.

(i) Genomic DNA: The genomic DNA is isolated from a donor. The desired gene is isolated from this DNA.

(ii) Vector: The vector which is generally a plasmid is used for the insertion of the desired gene.

(iii) Restriction endonucleases: These are the enzymes which are used for the fragmentation of the DNA. sing restriction enzymes such as endonucleases the DNA is fragmented. Endonuclease enzymes are known as molecular scissors as they produce nick in the DNA fragment at the desired place.

(iv) Ligase: This is the enzyme which is used for the ligation of the two fragments. The desired gene is introduced in the vector and attached with the help of ligase enzyme.

(v) Host: The recombinant vector is introduced into a competent host cell. The host cells are cloned or replicated for the production of more number of recombinant vectors which can be used for the formation of product.

What is the name of the sequence of base pairs which reads same on the two strands of DNA when orientation of reading is kept same?

The palindromic sequences are the sequences of the nucleotides which reads same from either side, that is forward and backward. These are the sequences which are present on the DNA or RNA double helix. The sequence is the same when reading from 5' to 3' on one strand and 3' to 5' on the other.

What is downstream processing?

Downstream processing is the recovery and purification of biochemical products with proper treatment. It is a series of events which includes cell separation, filtration, product recovery, extraction of product and purification and then treatment of product by chemical, physical and biological means. This is a necessary step in order to obtain a purified form of the product.

How to produce viruses as effective vectors?

In order to produce effective vectors, certain essential genes are spliced out from the viruses. This will render the virus harmless and allow space for the therapeutic genes to be inserted.
These new genes can be used for the treatment of certain genetic disorders. The viruses can also be used as vectors to transfer the genes to the plant or bacterial cells to produce certain important materials.

What is palindrome in DNA?

How does one visualize DNA on an agar gel?

Agarose gel electrophoresis is the method which is used to separate DNA, RNA and the proteins on the basis of their size. The technique uses the agar gel in which the DNA samples are loaded along with the dye ethidium bromide which is a fluorescent dye. The gel is placed under the source of UV light. The DNA forms separate bands according to their size. The larger one which runs to a smaller distance is placed at the bottom while the smaller ones which run faster are placed at the top.

With the help of a neat and labelled diagram describe steps in recombinant DNA technology.

The basic steps involved in the process of DNA technology are as follows.
$$(i)$$ The genomic DNA is isolated from a donor.
$$(ii)$$ Using restriction enzymes such as endonucleases the DNA is fragmented. Endonuclease enzymes are known as molecular scissors as they produce nick in the DNA fragment at the desired place.
$$(iii)$$ The fragments were taken out are then screened for the desired gene.
$$(iv)$$ Fragments of DNA with the desired gene are inserted into a cloning vector. Cloning vector can be a plasmid, cosmid or even a phage DNA. This insertion makes recombinant DNA or DNA (also known as chimeric DNA).
$$(v)$$ The recombinant vector is introduced into a competent host cell.
$$(vi)$$ These cells are later cultured to obtain multiple copies or clones of the desired fragment of DNA.
$$(vii)$$ These copies are used to transform suitable host cells which express the desired gene.

Explain the significance of 'palindromic nucleotide sequence' in the formation of recombinant DNA.

A palindromic sequence is a nucleic acid sequence on double-stranded DNA or RNA wherein reading 5' to 3' forward on one strand matches the sequence reading backward 5' to 3' on the complementary strand with which it forms a double helix. The significance of this sequence is that the sequences reads same in both the direction. This is important during the processes of replication, transcription and repair mechanisms which are directional.

Expand 'YAC' and mention what was it used for.

YAC refers to the yeast artificial chromosome which is derived from DNA of yeast S. cerevisiae by the process of genetic engineering. The DNA is ligated into the bacterial plasmid. By inserting large fragments of DNA, the inserted sequences can be cloned and mapped by chromosome walking and this method was used in the Human Genome Project. They can also be used for other cloning experiments.

Write the use of restriction endonuclease in the above process.

Recombinant DNA technology is the method which involves cutting and joining of different DNA fragments. This rDNA is then inserted into the host and allowed to produce the desired product. The restriction enzymes are the enzymes which cleave the DNA at specific recognition sites. This helps in the production of fragments with desired genes.

Figure representing the reactions associated with Polymer Chain Reaction (PCR).
Name the steps A, B, C in the progress.

Denaturation: The double-stranded DNA is heated up to 98$$^o$$ C which causes the hydrogen bonds to break and the two strands get separated.
Annealing: The two sets of primers are added which bind to the appropriate complementary segment of DNA strand.
Extension: The Taq polymerase enzyme polymerizes the nucleotide chain using the nucleotides provided in the medium and by using the template strand.

Answer in 40 to 80 words:
Explain in brief the separation and isolation of DNA fragments.

DNA can be separated by agarose gel electrophoresis in which agarose is used as a matrix. DNA sample is added to the slot and current is applied to it. The smaller molecules move faster and the larger molecules are retarded. In this method, separation is based on charge and size of the DNA fragment. Isolation of DNA from agarose gels by electrophoresis can be done using the DEAE-cellulose membrane. It is done by transfer of all DNA fragments from an agarose slab gel onto DEAE-cellulose paper. Individual fragments of DNA are then eluted using 0.1 M NaCl.

Given an example of the source of thermostable enzyme DNA polymerase.

Taq polymerase is an enzyme obtained from Thermus aquaticus which is a bacterium that normally lives in hot water springs. It is used in amplifying small quantity of short segments of DNA by polymerase chain reaction.

Use of a thermostable DNA polymerase from the bacterium, Thermus aquatics, made it possible to generate a billion copies of DNA in a very short time using a process. Name the process.

Polymerase chain reaction (PCR) is the process in which the amplification of the gene of interest is carried out with two sets of primers and a thermostable DNA polymerase enzyme Taq polymerase.

Define polymerase chain reaction.

Polymerase chain reaction or PCR is a technique used to amplify a single copy or a few copies of a piece of DNA to thousands of copies of a particular DNA sequence by using the thermo-resistant DNA polymerase.

Match the lists and find the correct option.

A-2,B-3,C-4,D-5

Solution : Colony hybridization is the selection of cells that contain the desired gene and transferring them into another medium. Separation of DNA fragments is done by the help of gel electrophoresis.Gradient centrifugation based on cell densities is used to purify DNA. Gene cloning in PCR or thermocycler is used to amplify a short stretch of DNA.

So the correct answer is "A-2,B-3,C-4,D-5"

Name two commonly used vectors in genetic engineering.

Vector is a DNA molecule used in genetic engineering as a vehicle to carry foreign genetic material from one cell into another cell where it is replicated or expressed. Examples of vectors include plasmid, cosmid, Lambda phages.

Describe the steps of PCR technique.

Polymerase Chain Reaction or PCR was developed by Karry Mullis in 1983. It is a technique to amplify DNA i.e. to make a large number of copies of DNA.

Requirements for PCR:

- Sample DNA (DNA which has to be amplified)

- Primers

- dNTPs (dATP, dTTP, dCTP, dGTP)

- Thermostable enzyme Taq Polymerase

- Thermocycler machine in which PCR has to be carried out.

Steps in PCR:

The PCR cycle has following three steps:

1. Denaturation: In this step, the two strands of double-stranded DNA are separated by heating to 94°C for 2 minutes. The two strands separate because the hydrogen bonds between the base pairs break.

2. Annealing: This step involves the binding of primers (short lengths of DNA about 20 bp long) to the 3' end of the single strands of DNA. The temperature of the reaction is lowered to 56°C so that the primers can anneal to the complementary sequences of the separated single strands of DNA.

3. Extension: In this step, the temperature of the reaction is raised to 72°C. The primers are extended by the enzyme Taq Polymerase. This enzyme is derived from the thermophilic bacteria Thermus aquaticus. This enzyme is stable at high temperatures even at 90°C. The enzyme adds nucleotides to the 3' end of the primers and extends them until a DNA strand which is complementary to the template DNA is formed.

Thus, one PCR cycle is completed and the amount of DNA doubles and so on.

Enlist the basic steps involved in recombinant DNA technology.

Recombinant DNA is the DNA that is composed of sequences that are derived from different sources. The process of recombination DNA technology consists of the following steps:

1. Isolation of genetic material (DNA)

DNA is enclosed within the membrane. So, to extract it out, the cell wall is broken down to release the genetic material. This can be done by using cell wall digesting enzymes such as Lysozymes for bacterial cells, cellulose for plant cells and chitinase for fungi cells.

2. Cutting of DNA at specific locations

This is done by using restriction enzymes that cut DNA at specific recognition sequences and give the desired DNA fragments.

3. Joining of DNA fragment

The DNA fragments generated with a cloning vector can be done by sticky ends ligation, blunt-end ligation and homopolymer tailing.

4. Insertion of DNA into the host cell.

The recombinant DNA can be inserted into the host cell by transformation, transduction, and vectorless gene transfer.

5. Selection and screening of transformed cells

The selection and screening of transformed cells can be by any of the following methods- immunological method, nucleic acid hybridization and blue-white screening or insertional inactivation.

List the key tools used in recombinant DNA technology.

Tools are enzymes, vehicle DNA, passenger DNA. Recombinant DNA technology involves the following steps are :

Isolation of DNA.
Fragmentation of DNA by restriction endonucleases.
Isolation of the desired DNA fragment.
Amplification of the gene of interest.
Ligation of the DNA fragment into a vector using DNA ligase.
Transfer of recombination DNA into the host.
Culturing the host cells on a suitable medium on a large scale.
Extraction of the desired product.
Downstream processing of the product as a finished product ready for marketing.

Differentiate between-: Gene cloning and cell cloning

Gene cloning is the technique to obtain clones or identical copies of a particular DNA molecule. Whereas incell cloning, a group of genetically.identical cells having descended from a single cell is obtained by recombinant DNA technology.

Very short answer type.
Selfing is a kind of inbreeding.

Selfing is a kind of inbreeding because the parents involved in selfing process have same genetic constitution. Moreover, selfing does not involve cross breeding.

What are molecular scissors ? Give one example. Explain their role in recombinant DNA technology.

Restriction endonucleases are also called as molecular scissors. Restriction endonucleases make random cuts in DNA, but the restriction endonucleases are site-specific.

Some examples of the restriction endonucleases are

Bam HI
Eco RI
Bgl II
Hae II
Hind II

Do you know what palindromes are?

The palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.

Write the methods to introduce foreign DNA into the host cells.

There are two ways of gene transfer by which foreign DNA can be introduced into the host cells:

1. Indirect gene transfer: When the foreign DNA is inserted into host cells by the use of a vector, the method of gene transfer is called indirect gene transfer. Indirect gene transfer generally occurs through:

(i) Plant tumours: This method has been used successfully to transform plant cells, which are difficult to transform. The gene is first inserted into the Ti plasmid of soil bacterium Agrobacterium tumefaciens. This method employs the usage of tumour causing microbe Agrobacterium tumefaciens to transfer DNA, by inducing the tumour formation in plants.

(ii) Viruses: The vector is first incorporated into a virus, which is then used to infect cells, carrying the foreign gene along with its own genetic material.

2. Direct gene transfer: The method of gene transfer in which DA is inserted directly into host cell is called direct gene transfer. Direct gene transfer occurs through:

(i) Heat shock: Cells are incubated with vector in a solution containing calcium ions at 0°C. The temperature is then suddenly raised to about 40°C. This heat shock causes some of the cells to take up the vector, as sudden heat develops pores in the membrane.

(ii) Electroporation: Cells are subjected to high-voltage pulse, which temporarily disrupts the membrane and allows the vector to enter the cell.

(iii) Gene gun: This technique fires microscopic gold or tungsten particles coated with foreign DNA at cells using a compressed air gun (burst of helium).

(Iv) Microinjection: A cell is held on a glass pipette with diameter much smaller than the cell, under a microscope and foreign DNA is injected directly into the nucleus using an incredibly fine micropipette, which punctures the plasma membrane.

Draw a neat labelled diagram of PBR322.

pBR322 is one of the standard cloning vectors obtained from E. coli. p stands for plasmid, BR stands for Boliver and Rodriguez and 322 is the order in which it was developed in the lab. It is genetically engineered and contains two antibiotic resistance genes for ampicillin and tetracycline, an origin of replication (ori), unique restriction sites for many restriction enzymes and a rop gene, which codes for proteins that are involved in regulating the copy number of plasmid inside the host cell.

"A very small sample of tissue or even a drop of blood can help determine paternity." Provide scientific explanation to substantiate the statement.

DNA from all cells of an individual shows the same degree of polymorphism and therefore becomes a useful identification tool.

Polymorphs are heritable and the child inherits 50% of the chromosome from each parent.

With the help of polymerase chain reaction (PCR), the small amount of DNA from blood can be amplified and be used in DNA fingerprinting to identify the paternity.

What is micro-injection?

Microinjection is a technique in which recombinant DNA is directly injected into the nucleus of an animal. In this, through a glass micropipette, foreign DNA is delivered directly into a living cell, oocyte or embryos of animal.

Why is the enzyme cellulase used for isolating genetic material from plant cells but not for animal cells?

DNA should be obtained in pure from for the action of restriction enzyme by treating the bacterial cell/ plant cell or animal tissue with enzyme.
Bacteria - Lysozyme; Plant cell - Cellulose; Fungus - Chitinase.

Describe briefly Chitinase.

During the isolation of DNA in processes of recombinant DNA technology, the fungal cell is exposed to an enzyme called chitinase. The chitinase enzyme dissolves the chitin cell wall of the fungus to open the cell for release of DNA along with other macromolecules such as RNA, proteins. polysachcharides and lipids.

Collect five examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base pair rules.

Palindromic nucleotide sequences in the DNA molecule are groups of bases that form the same sequence when read both forward and backward. Five examples of palindromic DNA sequences are as follows:
(1) $$5'$$ _____ $$A\,C\,T\,A\,G\, T$$ ______ $$3'$$
$$3'$$ _____ $$T\,G\, A\,T\,C\,A$$ ______ $$5'$$

(2) $$5'$$ _____ $$A\, A\, G\, C\, T\, T$$ ______ $$3'$$
$$3'$$ _____ $$T\, T\, C\, G\, A\, A\,$$ ______ $$5'$$

(3) $$5'$$ _____ $$G\, G\, A\, T\, C\, C$$ ______ $$3'$$
$$3'$$ _____ $$C\, C\, T\, A\, G\, G$$ ______ $$5'$$

(4) $$5'$$ _____ $$A\, G\, G\, C\, C\, T$$ ______ $$3'$$
$$3'$$ _____ $$T\, C\, C\, G\, G\, A$$ ______ $$5'$$

(5) $$5'$$ _____ $$A\ C\, G\, C\, G\, T$$ ______ $$3'$$
$$3'$$ _____ $$T\, G\, C\, G\, C\, A$$ ______ $$5'$$

Do eukaryotic cells have restriction endonucleases? Why?

No, the eukaryotic cells do not have restriction endonucleases. All the restriction endonucleases have been isolated from various strain of bacteria. Prokaryotes/bacteria have this enzyme as a defence mechanism to restrict the growth of bacteriophages.

Example-HindIII, EcoRII

Distinguish between plasmid DNA and chromosomal DNA.

Plasmid DNA is naked double stranded DNA that forms a circle with no free ends. It is associated with few protein but contains RNA polymerase enzyme. They are smaller than the host chromosome and can be easily separated.
Chromosomal DNA is double stranded linear DNA molecular associated with large proteins. This DNA exists in relaxed and supercoiled and provides a template for replication and transcription. It has free ends represented as

3′−5′3′−5′.

Explain briefly Restriction enzymes and DNA.

Two enzymes responsible for restricting the growth of bacteriophage in E.coli were isolated. One of these added methyl groups to DNA while the other cut DNA. The later was called restriction endonuclease. The restriction endonuclease binds to the DNA and cuts each of the two strands of the double helix at specific points in their sugar-phosphate backbones.

Draw a schematic sketch of pBR $$322$$ plasmid and label Ori and rop genes.

The labelled diagram of pBR322 plasmid is shown in the’ figure with.

(i)Eco RI and Bam HI as restriction enzymes

(ii) Ori and rop genes

(iii) amp (an antibiotic resistant gene)

draw-a-schematic-sketch-of-pbr322-plasmid-and-label-4

What are palindromic nucleotide sequences?

A palindromic sequence is a sequence of nitrogen bases in a double stranded DNA or RNA, which when read from 5' end to 3' end is same as that on the complementary strand read from 3'end to 5' end

example $$5'$$ _____ $$G\, A\, A\, T\, T\, C$$ _____ $$3'$$

$$3'$$ _____ $$C\, T\, T\, A\, A\, G$$ _____ $$5'$$

Why is a thermostable DNA polymerase needed in amplification/genetic engineering?

DNA polymerase is an enzyme, most of the enzymes function their best at body temperature. During the process of DNA amplification/genetic engineering ,PCR, the temperature may increase to around 60$$^o$$C, leading to denaturation of the enzyme. Therefore, a thermostable DNA pol is used during amplification

Example Taq DNA polymerase, obtained from Thermus aqaticus

What is elution in the process of separation of DNA fragments?

Elution is generally a process of extracting one material from another by washing with a solvent
It helps in the extraction of sample material into the solution so that it can be tested easily.
DNA is separated by the process of agarose gel electrophoresis.
When the sample DNA fragments are loaded on an agarose gel, being negatively charged it moves towards the anode.
The separated DNA fragments are stained with ethidium bromide and exposed to UV radiation.
The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution.
The DNA extracted from elution is amplified by PCR and can be used for several techniques like DNA fingerprinting etc.

Who discovered enzyme DNA polymerase?

DNA polymerase was discovered by Arthur Kornberg

Which enzymes are used for releasing macromolecules (DNA) from cell envelope?

Lysozyme for bacteria, cellulase for plant cell and chitinase for fungus.

What are the two sets of primers needed for amplification of gene?

The two sets of primers needed for amplification of a gene are

(i) Chemically synthesized short segments of DNA (oligonucleotides)
(ii) Enzyme DNA polymerase

Draw a schematic sketch of $$pBR 322$$ plasmid and label any two restriction sites.

The labelled diagram of pBR322 plasmid is shown in the’ figure with.

(i) Eco RI and Bam HI as restriction enzymes

(ii) Ori and rop genes

(iii) amp (an antibiotic resistant gene)

What is the best method for separation of DNA fragments?

The best method for separation of DNA fragments is by Agarose gel electrophoresis. Where DNA fragments are separated based on their size mobilized on a gel using electric current. This technique uses the negative charge on the DNA for segregating the segments

Name the scientists who generated first recombinant DNA molecules.

The first recombinant DNA molecules were generated in $$1972$$ by Paul Berg, Herbert Boyer, Annie Chang and Stanley Cohen

Expand PCR. Mention its importance in biotechnology.

Polymerase chain reaction (PCR) is a technique used to make numerous copies of a specific segment of DNA quickly and accurately. The polymerase chain reaction enables investigators to obtain large quantities of DNA that are required for various experiments and procedures in molecular biology, forensic analysis, evolutionary biology, and medical diagnostics.

The polymerase chain reaction is often considered as one of the most important scientific advances in the field of molecular biology. With this revolutionary yet inexpensive biochemical technology, it's possible to generate millions of DNA copies from a single strand of DNA.

How are restriction enzymes different from the topoisomerases functionally?

Restriction enzymes are nucleases which cut DNA into short pieces containing identifiable genes at specific sites. These pieces are then introduced into plasmids, yeast or plants cells.

Topoisomerase enzymes break and reseal strands of DNA which serve as starting points for replication.

What were the two main discoveries that led to the birth of genetic engineering?

(a) Discovery of restriction enzymes: These enzymes cut DNA into short pieces containing identifiable genes at specific sites. For example, Eco RI cut DNA at the sequence GAATTC/CTTAAG in double helical DNA.

(b) Presence of plasmid in bacteria

How does restriction endonuclease function.

Restriction enzymes or restriction endonuclease are used to cut DNA into smaller fragments. The cuts are always made at specific nucleotide sequences. the Different restriction enzymes recognize and cut different DNA sequences.

Why must a cell be made 'competent' in biotechnology experiments? How does calcium ion help in doing so?

It is necessary to make a cell competent in order to enhance the efficiency of the cell to take up foreign DNA easily.

When the cell is treated with a specific solution of divalent cation calcium. It increases the efficiency of the cell to take up the foreign DNA through the pores in the cell wall.

How is the amplification of gene sample of interest carried out using polymerase chain reaction (PCR) ?

The three steps are used for amplification

1. Denaturing - When the double-stranded template DNA is heated to separate it into two single strands.

2.Annealing - When the temperature is lowered to enable the DNA primers to attach to the template DNA

3.Extension - Polymerisation of nucleotide chain by the enzyme. Taq polymerase using the nucleotides provided in the medium.

How is DNA isolated from prokaryotic and eukaryotic cells?

(i) In prokaryotes, the bacterial cells are treated with certain lysozymes to break the cell wall.
(ii) In eukaryotes plant cell wall can be removed by treatment with cellulase; in case of fungi, it can be removed by treatment with chitinase.
Common steps of prokaryotes and eukaryotes:
After the boundary is broken, the nucleic acid (along with other compounds) is released from the cell. The DNA exists with certain proteins and RNA; so they have to be removed. Proteins are removed by treatment with proteases and RNA by treatment with ribonuclease. Other molecules are removed by appropriated treatment. Then DNA is precipitated out after the addition of chilled ethanol.

What are the functions of the enzymes isolated by Stewart Linn and Werner Arber?

The enzymes were responsible for restricting the growth of bacteriophage. One of the modification enzymes added methyl groups to the DNA. The second one, restriction endonuclease cut the DNA into segments.

State the role of 'biolistic gun' in biotechnology experiments.

To introduce alien DNA into host cells, suitable for plants, cells are bombarded with high-velocity microparticles of gold or tungsten coated with DNA molecules known as biolistic or gene gunplay important role in biotechnology experiments.

Draw a schematic sketch of pBR $$322$$ plasmid and label an antibiotic-resistant gene.

amp$$^R$$ marks the ampicillin resistant gene.
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What is PCR?

PCR - is Polymerase Chain Reaction carried in vitro or in the laboratory for amplification of DNA segments

Pathogen of which disease is best detected by PCR?

HIV or (AIDS virus) is best detected by PCR

What are the molecular scissors?

Restriction enzymes are also called 'molecular scissors' as they cleave DNA at or ear specific recognition sequences known as restriction sites. These enzymes make one incision on each of the two strands of DNA and are also called restriction endonucleases.

Why DNA cannot pass through the cell membrane? Explain. How is a bacterial cell made "competent" to take up recombinant DNA from the medium?

DNA is negatively charged particle and any charged particle cannot enter across the cell membrane. Once the DNA is induced in the vector cell it needs a host cell so that it can be cloned.

A bacterial cell is made competitive enough to take up the DNA because in normal condition cell does not take out the foreign gene. By treating the bacterial cell with a specific concentration of a divalent cation, such as calcium increases the efficiency with which DNA enters the bacteria through pores in the cell wall. Microinjection is another method in which DNA directly injected into the nucleus of an animal.

Who invented it?

PCR was invented by Kary Mullis in 1985. Polymerase Chain Reaction (PCR) is a technique for the amplification of DNA segments carried out in vitro.

Name the technique used for separation of DNA fragments.

The cutting of DNA by restriction endonucleases results in the fragments of DNA. These fragments can be separated by a technique known as gel electrophoresis.

What are the properties of ideal cloning vectors?

Characteristics of a cloning vectors

it must be small in size
It must be self-replicating inside host cell
It must possess restriction site for Restriction Endonuclease enzymes
Introduction of donor DNA fragment must not interfere with replication property of the vector
It must possess some marker gene such that it can be used for later identification of recombinant cell
it must possess multiple cloning site

Plasmid (PBR322):

PCR Machine/thermal cycler for PCR

The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). The Polymerase Chain Reaction (PCR) is an important tool for many applications. For example, it can be used to amplify a sample of DNA when there isn't enough to analyze (e.g. a sample of DNA from a crime scene, archeological samples) as a method of identifying a gene of interest, or to test for the disease.

In rDNA technology restriction endonucleases play a very important role. Give in brief its role in r- DNA technology.

Restriction endonuclease is a sub-class of nuclease enzyme which can digest phospho-diester linkage from non-terminal end of DNA. This enzyme is found in bacteria, it is part of a defence system of bacteria. It restricts replication of viral DNA within in bacterial cell by digesting Viral DNA at specific points. Specific points of digestion are located on different strands of DNA so, restriction fragments are always double-stranded.

What is DNA libraries?

DNA library or the gene library is the collection of DNA sequences from different organisms. These DNA sequences have been extracted, purified, sequenced and associated with a vector so as to use them for the purpose of analysis, comparative detection etc. Depending on the source of DNA, libraries can be of two types:

Genomic libraries- is a collection of the total genomic DNA from a single organism.
cDNA libraries- represents a collection of only the genes that are encoded into proteins by an organism. It is made using mRNA instead of DNA as the starting material.

How does one visualise DNA on an agarose gel?

The gel electrophoresis allows the separation of DNA fragments according to their size. The gel used is added with a dye called Ethidium Bromide (EtBr). The dye intercalates within the bases of the DNA fragments. After the process, the DNA fragments can be visualized under the UV light as the EtBr shows fluorescence under the UV light.

Explain the isolation of genomic (chromosomal) DNA (from bacteria/plant cell/ animal cell, by cell lysis).

Genomic (chromosomal) DNA from either bacteria, plant or animal cell can be isolated by lysis method. For plant cell or bacterial cell, first cell wall is mechanically disrupted. The cell wall is broken enzymatically using enzymes like lysozyme. After this, the cell membrane is disrupted using detergents and surfactants. The next step is precipitation of DNA. This step is done in order to separate DNA from the other cellular debris. In this step, sodium acetate is used to neutralize the negative charges on the DNA molecules. This makes the DNA more stable and less water soluble. After this, ice-cold isopropanol is added to precipitate DNA from the aqueous solution. The next step is purification step. Once the genomic DNA is released in the solution, phenol-chloroform solution is used to purify the sample. Phenol denatures the protein. After centrifugation, denatured proteins stay in the organic phase and DNA stays in the aqueous phase. The chloroform removes phenol residues from solution. The alcohol is added to remove any unwanted material and cellular debris. The purified DNA is then dissolved in the TE buffer.

A plasmid without a selectable marker was chosen as vector for cloning a gene. How does this affect the experiment?

A plasmid with a selectable marker (antibiotic resistance conferring gene) allows the selection of transformed or recombinant cells from the non-transformed or non-recombinant cells because in a population of cells, all the cells do not take up the plasmid. So, the selectable marker allows the transformed cell to live in the presence of antibiotics in the medium and non-transformed cell will perish. But without the selectable marker, the selection of the transformed cells will not be possible and in the presence of antibiotics, even the transformed cells will perish. Hence, it becomes a futile exercise.

Explain the vector pBR 322 with neat labelled diagram.

The pBR 322 vector is commonly used plasmid cloning vector in E. coli. In this, B and R stands for Bolivar and Rodriguez respectively. It is 4361 base pairs in length. Its molecular weight is 2.83 x 10$$^6$$ daltons. It contains origin of replication. It contains 2 antibiotic resistance genes, ampicillin and tetracycline. These antibiotic resistance genes are used as selectable markers. This is used to detect the transformants. It has specific restriction sites for 40 different restriction enzymes.

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A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium bromide, no DNA bands were observed. What could be the reason?

A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium bromide, no DNA bands were observed. This could happen due to one or all of the following reasons:

1. The mixture got contaminated with nucleases.

2. The concentration of the gel was not proper.

3. The anode and cathode were put in opposite orientation.

4. The concentration of EtBr was not enough to be intercalated within the bases of the DNA.

Explain the isolation of gene of interest(by electrophoresis).

Electrophoresis is a method of separating DNA fragment according to their size and molecular weight.

1. The cell containing gene of interest is taken.

2. The genome is isolated from the cell by lysis method.

3. The fragment of genome containing gene of interest is obtained using restriction endonuclease enzyme. The restriction enzyme cuts the DNA at a particular site.

4. Electrophoresis: The DNA fragment is then separated based on the size and molecular weight on electrophoresis. The samples are loaded in the well of the gel. One well is kept for a DNA ladder that contains DNA fragments of known lengths. After this, the gel is subjected to the electric current. The DNA molecules are negatively charged because of the phosphate groups in the sugar-phosphate backbone. Hence, they will move through the matrix of the gel towards the positive pole. The large fragment of DNA will be near the negative pole and smallest fragments of DNA will be near the positive pole.

5. Once, the gene of interest is separated on electrophoresis, southern blotting is carried out. In this, the DNA is transferred to the nitrocellulose paper. It is then hybridized with the radioactive probe and seen using X-ray.

What is gel electrophoresis?

Electrophoresis is defined as a technique of separation of the charged molecules of the substance in a solution, by subjecting them to an electric field. The technique helps to determine the number, amount and the mobility of the components in a given sample or separate them. The resolving power of electrophoresis was greatly improved by the introduction of a technique called zone electrophoresis. A homogenous inert supporting medium such as paper, cellulose acetate, polyacrylamide or agarose gel, etc. was used to support the sample solution instead of using it freely as in the earlier case. In this technique, the isolated components form distinct zones on the supporting medium and hence the name. The zone electrophoresis performed using gel is referred as "Gel Electrophoresis". It can be carried out in any one of the two modes viz., horizontal and vertical electrophoresis.
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Describe selection and selfing of superior recombinants.

The progeny observed for the desirable combination of characters. Such plants are selected and carefully observed and scientifically evaluated for the success of breeding objective. The plants which are superior to both the parents and exhibit hybrid vigour are collected. Such plants are self pollinated for few successive generations to get homozygosity. Due to this plant attains a state of uniformity and characters do not segregate in the progeny.

Why DNA has fragments in band 'D' moved farther away in comparison to those in band 'C'?

The given image shows the process of gel electrophoresis of DNA in which the fragments get separated on the basis of their size. DNA fragments are negatively charged so, all move towards anode. Smaller particles move faster and farther away from the start, along the gel, in response to the electrical field (current) supplied to the gel.

So, it can be inferred that being smaller in size, D fragments move farther away in comparison to those in band C

Explain the three steps involved in each cycle of polymerase chain reaction.

1) Denaturing stage

Amid this stage, the mixed drink containing the format DNA and the various center fixings is warmed to 94-95⁰C.

The high temperature causes the hydrogen bonds? between the bases in two strands of layout DNA to break and the two strands to isolate.

This outcome in two single strands of DNA, which will go about as layouts for the generation of the new strands of DNA.

It is critical that the temperature is kept up at this phase for enough time to guarantee that the DNA strands have isolated totally.

This generally takes between 15-30 seconds.

2) Annealing stage

Amid this stage, the response is cooled to 50-65⁰C. This empowers the preliminaries to connect to an explicit area on the single-stranded format DNA by a method for hydrogen holding (the correct temperature relies upon the dissolving temperature of the ground works you are utilizing).

Preliminaries are single strands of DNA or RNA. the grouping that is around 20 to 30 bases long.

The preliminaries are intended to be integral? in arrangement to short areas of DNA on each finish of the grouping to be duplicated.

Preliminaries fill in as the beginning stage for DNA blend. The polymerase catalyst can just add DNA bases to a twofold strand of DNA. Just once the preliminary has bound can the polymerase protein append and begin making the new corresponding strand of DNA from the free DNA bases.

The two isolated strands of DNA are corresponding and kept running in inverse ways (from one end - the 5' end – to the next - the 3' end); therefore, there are two groundworks – a forward preliminary and a switch preliminary.

This progression, as a rule, takes around 10-30 seconds.

3) Extending stage

Amid this last advance, the warmth is expanded to 72⁰C to empower the new DNA to be made by a unique DNA polymerase chemical which includes DNA bases.

DNA polymerase is a chemical taken from the warmth cherishing microscopic organisms.

This microscopic organism typically lives in hot springs so can endure temperatures above 80⁰C.

The microbes DNA polymerase is truly steady at high temperatures, which implies it can withstand the temperatures expected to break the strands of DNA separated in the denaturing phase of PCR.

DNA polymerase from most different living beings would not have the capacity to withstand these high temperatures, for instance, human polymerase works in a perfect world at 37˚C (body temperature). 72⁰C is the ideal temperature for the Taq polymerase to fabricate the corresponding strand. It appends to the preliminary and after that adds DNA bases to the single strand one-by-one in the 5' to 3' course.

The outcome is fresh out of the plastic new strand of DNA and a twofold stranded atom of DNA.

The term of this progression relies upon the length of DNA grouping being intensified yet more often than not takes around one moment to duplicate 1,000 DNA bases (1Kb).

These three procedures of warm cycling are rehashed 20-40 times to create bunches of duplicates of the DNA arrangement of intrigue.

The new sections of DNA that are made amid PCR additionally fill in as layouts to which the DNA polymerase protein can connect and begin making DNA. An outcome is a colossal number of duplicates of the explicit DNA fragment delivered in a moderately brief timeframe.

Name the substance used as the medium / matrix in gel electrophoresis. Mention its source.

Agarose is a polysaccharide, generally extracted from certain red seaweed. Agarose is frequently used in molecular biology for the separation of molecules, especially DNA, by electrophoresis.

With help of diagram explain plasmid BR322.

pBR322 is a plasmid and was one of the first widely used E.coli cloning vectors.
p stands for plasmid and BR stands for Bolivar and Rodriguez.
pBR322 is 4361 base pairs in length and has two antibiotic resistance genes- the gene Amp^r encoding the ampicillin resistance and the gene tetA encoding tetracycline.
It contains the origin of replication (Ori).

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What is gel electrophoresis ? Explain how DNA fragments are separated and detected using this.

Gel electrophoresis is a technique used to separate components of a mixture on the basis of their size. The mixture could be a DNA, an RNA or even a mixture of proteins. A gel matrix of suitable material (e.g., agarose gel) is spread upon the electrophoresis plate. The mixture is loaded on to a grove in the gel and an electrical field is applied to it. The particles of the mixture move away from the start towards the oppositely charged end based on their molecule size and charge. The particles with a smaller size have been reported to move faster and farther away from the start.
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___________ is the revolutionary event in biotechnology after cloning.

Recombinant DNA technology is the revolutionary event in biotechnology after cloning.

Recombinant DNA technology involves joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry.

Write the two components of the first artificial recombinant DNA molecule constructed by Cohen and Boyer.

Restriction enzyme
Vector

What are plasmids ? Mention any two features of an ideal plasmid.

A plasmid is a small, circular DNA molecule that is different from the cell’s chromosomal DNA. It can replicate independently of the chromosomal DNA and are found in most of the bacteria. Plasmids are generally used as cloning vectors. For being a good cloning vector, a plasmid must have the following characteristics:

• An origin of replication or ori from where the replication begins.

• A selectable marker that usually has a specific action like antibiotic resistance that enables it to grow on some selective media.

• It should itself be a small fragment of DNA that is easy to ligate in a different strand of DNA.

• It should have a high copy number.

What is meant by gene gun method?

The gene gun method is a method used for genetically modifying plants. With the use of a gene gun, the gene gun method delivers extra DNA directly into a plant’s nucleus. The method is also commonly called particle acceleration or microprojectile bombardment.
The gene gun can be used on seedlings or tissue culture cells. Prior to injecting the DNA into the plant tissue via the gene gun, either microscopic gold or tungsten particles are liberally coated with many hundreds of copies of genes. The particles are then forced into the nucleus with the gene gun.
The gene gun has revolutionized the genetic modification of plants. The first gene gun method of DNA modification was developed in 1980.
The process relied on a 22-caliber cartridge to shoot the gene-coated metal particles into the plant’s cell. Nowadays, modern versions of the gene gun operate differently.
The modernized gene gun depends on a vacuum chamber and high-pressure gas to hurl the metal particles into the plant cells.
The gene gun is highly effective at modifying the DNA and genetics of plant cells.

Give reasons for the following:
Restriction enzymes are called chemical scalpels.

The DNA is cleaved by a restriction enzyme at or near specific recognition sequence known as Restriction sites. The incision is made by this restriction enzyme on two strands of DNA. Therefore they are also called as Molecular scissors and also restriction endonucleases.

Describe the steps in rDNA technology.

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Write any four ways used to introduce a desired DNA segment into a bacterial cell in recombined technology experiments.

Four ways to introduce the desired DNA segment into the bacterial host are:

Heat shock based transformation
Electroporation
Using disarmed bacteriophages as vectors (Transduction)
Micro-injection.

Which of the following is not component of yeast artificial chromosome?

Yeast artificial chromosomes are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces, which is then ligated into a bacterial plasmid.
By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking.
The primary components of a YAC are the ARS, centromere, and telomeres from Saccharomyces.
Additionally, selectable marker genes, such as antibiotic resistance and a visible marker, are utilized to select transformed yeast cells.
Without these sequences, the chromosome will not be stable during extracellular replication, and would not be distinguishable from colonies without the vector

A. What is the role of Cry I Ac, Cry IIAb and Cry I Ab?
B. (i) Write the sequence of restriction site of EcoRI and give the sequence of sticky ends after EcoRI digestion
(ii) Name the source of EcoRI restriction enzyme

A. Cry I Ac, Cry II Ab genes are used control cotton bollworm and Cry I Ab is used to controlling corn borer.

B. (i) the sequence of restriction site of EcoRI is G'AATTC and EcoRI creates 4 nucleotide sticky ends with 5' end overhangs of AATT.

(ii) EcoRI source is bacteria E-coli of strain RY13.

Given is the diagram of agarose gel kept under UV light.
How are the separated DNA fragments finally isolated?

The DNA fragments are isolated by a process called as elution. In this process, the visible fragment of desired size ( clearly visible as an orange band under UV illumination) is cut out from Gel using scalpel or blade, after which various chemical procedures are followed to separate out the DNA band from the solidified gel.

Explain how $$rDNA$$ gets introduced into a plant cell.

The second delivery method is a “gene gun,” which fires gold particles carrying the foreign DNA into plant cells. Some of these particles pass through the plant cell wall and enter the cell nucleus, where the transgene integrates itself into the plant chromosome.

Mention two uses of cloning vectors.

There are many types of cloning vectors, but the most commonly used ones are genetically engineered plasmids. Cloning is generally first performed using Escherichia coli, and cloning vectors in E. coli include plasmids, bacteriophages (such as phage λ), cosmids, and bacterial artificial chromosomes (BACs).

Mention the significance of Gel-electrophoresis in r-DNA technology.

Gel electrophoresis is a technique used in recombinant DNA technology for the separation of fragments of DNA on the basis of their size. In this method the DNA fragments are run in an electric field and because the fragments have the same charge by mass ratio the separation is only on the basis of the size of the fragments, with the smaller fragments moving faster (with the bands being seen lower in the gel) and the larger fragments slower (with the bands being seen at the top). In recombinant DNA technology, the isolated DNA fragments can be separated and analysed by this technique.

Study the diagram given and answer the question.
What does E represent in the diagram?

Fragment E represents undigested complete DNA that is larger in size in comparison to all the other fragments in the gel. If fragment E would have been digested other bands would have been seen in the same lane along with band E. Since, this band is present towards the cathode, it has not moved far in the gel and is large in size.

Name two commonly used vectors in rDNA technology.

Two commonly used vectors in rDNA technology are:

Plasmid vectors: Extra-chromosomal, self-replicating, double-stranded circular DNA molecules found in various bacteria and in some yeast.
Bacteriophage vectors: Viruses that infect bacterial cells by injecting their DNA into these cells.

Study the diagram given and answer the question.
Why have DNA fragments in band 'D' moved farther away in comparison to those in band 'C'.

In gel-electrophoresis the DNA fragments move according to their size, with the smaller fragments travelling further in the gel and the band appearing lower in comparison to larger DNA fragments that move slower in the gel and cannot move far. As a result of this, the band formed of such large DNA fragments appear at the top (i.e near the cathode). Hence, movement of fragment D further in the gel indicates that it is formed of smaller fragments and the fragment C that does not move far is formed of larger sized DNA fragments.

Study the diagram given and answer the question.
Identify the anode end in the diagram.

DNA is negatively charged and therefore attracted towards the positively charged end and therefore it moves from the cathode (the negatively charged end) towards the anode (positively charged end) in an electric field. In the given diagram the DNA fragments move from the end A towards the end B in the gel and hence the end B is the anode end.

Restriction enzymes are considered as a type of endonuclease. Why?

An endonuclease is an enzyme that cleaves a DNA fragment by separating the nucleotides within the fragment (by breaking the phosphodiester bonds) and not the end ones. The restriction enzyme also cleaves the DNA at specific sites called restriction sites (recognition sites) located within the molecule. Hence, restriction enzymes are also considered a type of endonuclease.

REN are called as molecular scissors. Why?

Restriction enzymes can cut the DNA into fragments at specific sites known as the restriction sites. These restriction sites (recognition site) are unique sequences of 4 or 6 base pair length. E.g: the restriction enzyme called EcoRI cuts the DNA fragments at the site having the sequence GAATTC.

Give brief account of replication of bacteriophages.

After infection, phage DNA forces the host to work for the parasite, so that complete host machinery is utilized for replication of phage particle. The infected cell thus creates new protein subunits for the phage head and tail and also makes additional DNA segments identical to DNA of the invading phage particle.

What are molecular scissors? Where are they obtained from?

Restriction enzymes that are used to cutting the DNA into pieces. They cleave DNA at or near specific recognition sequences known as restriction sites. Thus they are called as molecular scissors. Usually, they are some proteins produced by bacteria. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms

Describe the steps that constitute one cycle of reactions in PCR.

Step 1: Denaturation

In this process, the two strands in the DNA double helix needs to be separated like in DNA replication. This separation takes place by increasing the temperature of the mixture. As a result, the hydeogen bonds between the complementary DNA strands break. This step is called denaturation.

Step 2: Annealing

In this process, primers bind to the target DNA sequences and it initiates polymerisation. This can occur only when the temperature of the solution is low. One primer binds to each strand.

Step 3: Extension

New strands of DNA are synthesized using the original strands as templates. The enzyme DNA polymerase joins thee free DNA nucleotides together. This enzyme is also called Taq polymerase and it is isolated from a thermophilic bacteria called Thermus aquaticus. The sequence of nucleotides in the original (template) DNA strand determines the order in which the free nucleotides are added.

One cycle of PCR results in the formation of two double-stranded sequences of target DNA, in which each contains one newly made strand and one original strand.

Define PCR.

PCR is known as Polymerase Chain Reaction. It is a widely used laboratory technique to generate many copies of a particular region of DNA.

Expand PCR. How it helps in amplification of gene of interest ?

Polymerase chain reaction (PCR) is a method widely used in molecular biology to make many copies of a specific DNA segment. Using PCR, a single copy (or more) of a DNA sequence is exponentially amplified to generate thousands to millions of more copies of that particular DNA segment. The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.
The basic steps are:

Denaturation (96°C): Heat the reaction strongly to separate, or denature, the DNA strands. This provides a single-stranded template for the next step.
Annealing (55 - 65°C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.
Extension (72°C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.

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Name the vectors which can be used for transfer of genes to plant and animal cell?

Plasmids are the common vectors for both plants and animals. But there are also some other vectors like-

In plants Agrobacterium tumifaciens, a pathogen of several dicot plants is able to deliver a piece of DNA known as 'T-DNA' to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.

And in animals retrovirus have the ability to transform normal cells into cancerous cells.

Explain any two methods for introduction of recombinant DNA into host cell.

Electroporation: This method is based on the use of the short electrical pulses of high field strength. Electroporation causes the uptake of DNA into protoplasts by temporary permeabilization of the plasma membrane to macromolecules. Protoplasts and foreign DNA are placed in a buffer between two electrodes and a high-intensity electric current is passed, the alternating current of about 1 MHz is applied to align the protoplast by dielectrophoresis. Once aligned, fusion is induced by applying one or more direct current pulses (1-3kV /cm, 10-100 (is), then the alternating field is reapplied briefly to maintain close membrane contact for fusion (Fig. 16.1). Electric field damages membranes and creates pores in membranes. DNA diffuses through these pores immediately after the electric field is applied, until the pores are resealed. The technique is optimized by using appropriate electric field strength (defined as the applied voltage divided by the distance between two electrodes).
Liposomes have also been used as a carrier for the introduction of nucleic acid into plant protoplasts. Liposomes are small lipid sacs containing plasmids and are prepared artificially. The fusion of liposomes with plant protoplasts is stimulated by chemicals such as PEG (endocytosis). Liposomes mediated transformation has been achieved by including positively charged agents such as cations in the transformation mixture or using the cationic liposome preparation.

'The basis of genetic engineering is the discovery of the fact that genes can be cut and joined with the help of enzymes."
a. Name the enzyme used for cutting genes.
b. Name the enzyme used for joining the genes.

a. Restriction Endonuclease/ Genetic Scissors
b. Ligase/Genetic Glue

Describe the process of amplification of ''gene of interest '' using PCR technique.

Polymerase chain reaction (PCR) is the process in which the amplification of the gene of interest is carried out with two sets of primers and a thermostable DNA polymerase enzyme Taq polymerase. The process involves the following steps:

Denaturation: The double-stranded DNA is heated up to $$94^oC$$ which causes the hydrogen bonds to break and the two strands get separated.
Annealing: The two sets of primers are added which bind to the appropriate complementary segment of the DNA strand at $$54^oC$$.

Extension: The Taq polymerase enzyme polymerizes the nucleotide chain using the nucleotides provided in the medium and by using the template strand at $$72^oC$$.

Describe the formation of recombinant DNA by the action of EcoRI.

Restriction endonuclease recognizes a specific palindromic nucleotide sequence in the DNA.

The recognition site for EcoRI

5' - G A A T T C - 3'

3' - C T T A A G - 5'

Restriction enzymes cut the strand of DNA a little away from the center of the palindromic sites, but between the same two bases on the opposite strands:

The enzyme cuts both the vector DNA and the foreign DNA at the same site. EcoRI cuts the DNA between bases G and A only when the sequence GAATTC is present in the DNA.
This leaves single-stranded portions at the end which are overhanging stretches called sticky ends.
Sticky ends form hydrogen bonds with their complementary cut counterparts. This stickiness of the end facilitates the action of enzyme DNA ligase.
Thus Recombinant DNA is formed.

Mention application of PCR in the field of Biotechnology

Write the palindromic nucleotide sequence EcoRI recognises.

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Observe the table in which size of different DNA fragments are given and answer the questions:
DNA fragments $$A$$ $$B$$ $$C$$
Size (in Base Pairs) $$700$$ $$1500$$ $$3000$$
Explain the process of separating the DNA fragments.

Gel electrophoresis is the technique used to separate DNA fragments as per molecular weight.
Gel electrophoresis is basically the process by which we take the DNA, and run an electric charge through it. The DNA, being negatively charged by default, will move towards the positive side. As this happens, the DNA with lower molecular weight will move faster, as there will be less hindrance caused by the mesh-like structure, to the small DNA particle as compared to a large DNA molecule.

"DNA copies generated will be similar, but may not be identical to the original." Explain.

The DNA copying mechanism, also called the DNA replication processes, are highly precise, but they are not completely accurate. Each time when the DNA is copied, some changes or mistakes might take place in formation of the new DNA strand. These mistakes account for the variations that occur amongst the individuals in a population. In sexual reproduction, these changes increase in rate of occurrence due to involvement of two individuals with different patterns of accumulation of variations. The variations hence created, are unique in nature. Thus, the DNA copies generated are similar, but not identical to the original.

Observe the table in which size of different DNA fragments are given and answer the questions:
DNA fragments $$A$$ $$B$$ $$C$$
Size (in Base Pairs) $$700$$ $$1500$$ $$3000$$
In the process of separating DNA fragments which fragements moves faster?

The DNA fragments can be isolated using the technique called Agarose gel electrophoresis. In this technique, the DNA fragments are present under the electric field and attracted towards one terminal (i.e., positive terminal) of the apparatus, as the DNA is negative in charge, the DNA moves towards the positive terminal. Here, the DNA fragments move inside a mesh-like structure of agarose gel which limits the movement of the DNA fragments. Thus, in this process of separating DNA fragments, the shorter element moves faster.

Hence, A>B>C is the correct order of the speed of fragments.

Mention application of PCR in the field of Diagnostics

PCR in the field of Diagnostics is used in different ways, such as following: -
Detecting viral disease infection,

Detecting cancer markers,

Detecting Genetic markers for diseases, etc.

All cloning vectors do have a 'selectable marker'. Describe its role in recombinant DNA-technology.

Role of Selectable markers in recombinant DNA technology: -

They help in selecting the host cells which contain the vectors (transformants)
They eliminate the possibility of selecting the non-transformants instead of transformants.
Generally, the genes encoding resistance to antibiotics (e.g. tetramycin, ampicillin, etc.) are useful selectable markers for E.coli.

Draw the vector DNA and a foreign DNA showing the sites where EcoRI has acted to form the sticky ends.

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What is the source organism for EcoR1, restriction endonuclease?

E.coli - is the source organism for restriction endonuclease EcoR1.

What does 'competent' refer to in competent cells used in transformation experiments?

The ability of the bacterial cell to take up DNA through pores in cell membrane is called competence. DNA is a hydrophilic molecule and hence, it cannot pass through the cell membrane. So, cell is made 'competent' by exposing them to an appropriate chemical or electric charge or heat shock. Example, of chemical is Calcium Chloride.This process is referred to as transformation.

While doing a PCR, 'denaturation' step is missed. What will be its effect on the process?

$$\textbf{Explanation:}$$

$$\bullet$$ In the denaturation step, the target DNA is heated to a high temperature (usually $$94^0$$C), resulting in the separation of the two strands.

$$\bullet$$ Each single strand of the target DNA then acts as a template for DNA synthesis.

$$\bullet$$ During PCR if the initial denaturation step is missed, the dsDNA for which the polymerization process has to be done will not split into two template strands, and as a result primer annealing would not occur. Consequently extension would also not take place and we would not observe amplification of DNA.

What is the significance of adding proteases at the time of isolation of genetic material (DNA).

Proteases helps in removing protein during the process of obtaining pure DNA. Other macromolecules are eliminated with the help of suitable enzymes during this process. At the end, pure DNA is isolated.

Restriction enzymes should not have more than one site of action in the cloning site of a vector. Comment.

Restriction enzymes are endonucleases that cleave the phosphodiester bonds of the DNA backbone at specific sites. This helps in isolation and insertion of DNA into the plasmid of the recipient organism or the cloning vector. They should not have more than one site of recognition as it will interfere with the functioning of other genes present on the plasmid of the cloning vector.

A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium bromide, no DNA bands were observed. What could be the reason?

$$\textbf{Explanation:}$$

Following are the possible reasons:

$$\text{a)}$$ The gel plate may not be exposed to UV light. Ethidium bromide intercalates with DNA and further give flourescence only when exposed to UV light.

$$\text{b)}$$ Electrodes may have been put in the wrong orientation; with anode or positive electrode towards the loading well.

$$\text{c)}$$ As DNA is negatively charged, It move towards the positive electrode. When the anode or positive electrode is near the loading well, separated DNA may move out of the gel.

$$\text{d)}$$ The sample may be contaminated with protein or RNA.

$$\text{e)}$$ Improper loading of the sample in the wells.

Restriction enzymes that are used in the construction of recombinant DNA are endonucleases which cut the DNA at 'specific-recognition sequence'. What would be the disadvantage if they do not cut the DNA at specific-recognition sequence?

Specific-recognition sequence in a DNA provide sticky ends at which recombination of genes takes place. This further leads to replication of selected gene. If endonudease fails to cut DNA at specific-recognition sequence; then recombination or replication will fail to take place.

Taking examples under each category, discuss upstream and downstream processing.

Upstream processing: This involves identifying a material which can be transformed for making the final product. For example, in the process formation of alcohol by Yeast, the raw material like grape juice is added to the fermenter, measured quantities of yeast culture is added, the temperature, pH and concentration of the mixture is brought to optimum. This results in formation of alcohol after a stipulated period of time. This is called upstream processing.

Downstream processing: This involves suitable purification and isolation of final product. Let us take that example of fermentation to understand this. Purification of alcohol through distillation is part of downstream processing.

When a foreign DNA is introduced into an organism, how is it maintained in the host and how is it transferred to the progeny of the organism?

When a foreign DNA is introduced into an organism; it is called a genetically modified organism or transgenic organism. It is inserted with the help of vectors into the recipient organism. It then becomes an integral part of the host's genome like other indigenous genes, due to recombination. Usually the recombinant organisms are made to reproduce asexually, through technique like tissue culture to preserve the recombinant trait in the offsprings.

Who is credited for developing the recombinant DNA Technique (RDT)?

Stanley Cohen, Herbert Boyer et al (1973) are credited for developing the recombinant DNA technology.

How does one visualise DNA on an agarose gel?

$$\textbf{Explanation:}$$

$$\bullet$$ Agarose matrix is the most commonly used matrix nowadays and agarose is a polysaccharide extracted from sea weed.

$$\bullet$$ DNA fragments separate according to size through the pores of agarose gel. The agarose gel is mixed with ethidium bromide during the casting process. Ethidium bromide has a tendency to intercalate with DNA.

$$\bullet$$ The separated fragments of DNA can only be seen after staining with ethidium bromide (EtBr) followed by exposure to UV radiation, DNA can be observed in the form of bright orange colored bands. EtBr shows fluorescence when exposed to UV light.

What is meant by gene cloning?

A set of experimental methods used to assemble recombinant DNA molecules and to use them for cloning in host organism Is called gene cloning. Gene cloning Involves following main steps:
$$*$$ Selection of a suitable donor.
$$*$$ Treating the donor DNA with enzymes to obtain small fragments.

$$*$$ Isolating the required fragment by gel electrophoresis
$$*$$ Transferring the fragment in a suitable host.

A plasmid without a selectable marker was chosen as vector for cloning a gene. How does this affect the experiment?

$$\textbf{Explanation:}$$

$$\bullet$$ Some genes called selectable markers help in selecting those host cells which contain the vectors (transformants) and eliminating the non-transformants.

$$\bullet$$ Transformation is a process through which a piece of DNA is introduced in a host bacterium.

$$\bullet$$ In the absence of a selectable marker, it will not be possible to differentiate between transformants and non-transformants.

$$\bullet$$ Thus, carrying the experiment to its logical end would be impossible in the absence of a selectable marker.

Describe the role $$CaCl_2$$ of in the preparation of competent cells?

PCR is a useful tool for early diagnosis of an infectious disease. Elaborate.

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What are Cosmids?

Cosmids are hybrid of plasmid and 2 (Lambda) phage. The plasmids, in which DNA sequence of 'Cos' site of λ (Lambda) phage are inserted, are known as cosmids.

What do you understand by molecular probes? Explain its utility.

Molecular Probes:
Segments of DNA or RNA with the help of which cDNA or RNA segments of any organism present in it can be identified are called molecular probes.
The molecular probes are of two types.

DNA probes.
RNA probes.

Importance of Probes:

These are used to identify specific DNA segments used in the research of genetic engineering.
Pollutants in food can be detected with the help of probes.
These are used in the field of forensic science, in resolving disputed paternity issues and establishing the family relationship.
These can be used to identify an improved variety of crops and hybridized seeds of crops.

What are cloning vectors?

In recombinant DNA technology, in order to introduce the desired gene in the targeted plant/animal, a carrier is required which can carry the desired gene and can enter the targeted plant/animal and replicate it's DNA. This carrier is called a vector. Plasmids, Bacteriophages and Cosmids are used as a vector in recombinant DNA technology.

Describe different blotting techniques in detail.

Blotting Technique:
In this technique, DNA segments are derived on agarose gel by electrophoresis and are then transferred and stabilised on a nitrocellulose filter. These are then identified by hybridization with DNA probes. This process is called the blotting technique.

1. Southern Blotting Technique: E.M. Southern (1975) first used this method and transferred DNA segments on the nitrocellulose filter. This technique was named a Southern blotting technique. By this technical analysis of DNA, segments are done.

2. Northern Blotting Technique:
Alwin et al (1979) transferred RNA segments after electrophoresis on amino benzyl oximetry (OBM) membrane in place of nitrocellulose filter. This technique was named as Northern blotting technique. This technique is used for the analysis of RNA segments.

3. Western Blotting Technique:
Tobin et al. (1979) first break down proteins into polypeptides with the help of sodium-do-decyl sulphate (SDS). After separating them with the help of electrophoresis, transferred these on a nitrocellulose filter or nylon membrane. These were then decoded on an X-ray plate and identified the protein. By this technical analysis of proteins is done. This is called the western blotting technique.
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1864836_1873272_ans_5642a893c2914380a2dbe889c7f55954.jpg

Define the restriction endonuclease enzyme.

The enzyme which cut the DNA molecule at specific sites is called restriction endonuclease. These enzymes are just like molecular scissors, which cut DNA into the segment at specific sites.

Write the names of any three methods of direct gene transfer.

Three methods of direct gene transfer are;
Gene gun method
Electroporation
Lipofection
Microinjection

Write a short note on the importance of Bacteriophage as a clonal vector.

Bacteriophages are a better vector than the plasmids due to the following reasons:

It can clone the DNA segment of a relatively large size (24 kbp).
Every bacteriophage produces one plaque area in the culture through which testing is comparatively easy.

λ (Lambda) phage is more widely used as a vector than $$M_{13}$$phage because λ phage is having linear and double-stranded DNA and it is a phage of E.coli and large-sized foreign DNA can be easily inserted in it's DNA.

Name the gels used in the gel electrophoresis technique.

The gels used in gel electrophoresis techniques are
Agarose gel.
Polyacrylamide gel.

Define recombinant DNA technology?

Various effective measures required to incorporate changes in the DNA make up of any organism is called recombinant DNA technology.

What do you understand by indirect gene transfer?

When gene transfer is done through some biological vector, the process is called the indirect method. In this process, the desired gene is first transferred in the vector and then through the vector, it is transferred into the target organism. Hence, such a transfer of gene is called indirect gene transfer.

Explain the microinjection technique of gene transfer.

Microinjection:
In this method, genes are injected in plant protoplasts or cells with the help of a glass needle of 0.5 - 1.0 mm diameter or micropipette directly in the cytoplasm or nucleus of the protoplasts. This is a suitable method of transferring genes in isolated protoplasts.
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Write an illustrated account of physical methods of gene transfer.

Physical methods of gene transfer are as follows:

Gene gun method
Electroporation
Lipofection or Liposome mediated gene transfer.
Microinjection

1. Gene gun: Gene gun is also known as particle gun, shotgun or microprojectile, etc. By this technique, gene transfer is possible in the intact plant cells. (cells having intact cell wall). This technique of gene transfer was first used by Klein and Co-workers (1987) in onion cells for transferring DNA and viral RNA.
In this process, the minute gold or tungsten particles of 1-3 mm diameter (microparticles) coated with desired DNA are shot (bombarded) in the target cells with the help of a microprojectile.
These desired DNA coated gold or tungsten particles penetrate the cell wall and enter the cell where desired DNA incorporates with the host cell DNA and forms transgenic DNA. By the use of this method, gene transfer has been successfully achieved in wheat, rice, maize, tobacco, and soybean, etc. This technique is being used now in all types of plants worldwide.

2. Electroporation: In this technique of gene transfer, the targeted protoplasts, plant cells, or tissues are subjected to pulse of high voltage. As a result, minute temporary pores are formed in the plasmalemma (plasma membrane).
The desired DNA enters through these minute pores. The target cells or the tissues are kept in a solution containing the desired DNA and then subjected to a high voltage pulse. The DNA from the solution first enters into the cells and then gets incorporated into the genome of the cells. This method is extensively used for transferring genes in the cells of monocotyledonous plants.

3. Liposome mediated gene transfer: In this method of gene transfer, spherical lipid molecules filled with desired DNA and water are used for transferring genes. These DNA containing lipid capsules first stick to the plasmalemma (plasma membrane) and then fuse with it.
The DNA present in these first enters into the cell and then enters the nucleus where it gets incorporated in the genome of the host cell. The liposome directed gene transferring technique also called as lipofection technique is a highly effective technique for transferring genes in bacteria, animal & plant cells.

4. Microinjection: In this method, genes are injected in plant protoplasts or cells with the help of a glass needle of 0.5 - 1.0 mm diameter or micropipette directly in the cytoplasm or nucleus of the protoplasts or cells of plants. This is a suitable method of transferring genes in isolated protoplasts.
Note: Besides the above methods of gene transfer other methods are also employed for transferring genes. These are Ladder dependent gene transfer and Silicon carbide fibre dependent gene transfer.
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What is accomplished during PCR amplification?

PCR stands for a polymerase chain reaction and is used to produce a multitude of copies (amplification) of a desired piece of DNA. By using PCR, 1 billion copies are produced from a single DNA fragment.

Addgene: What is Polymerase Chain Reaction (PCR)

These are important set of enzymes used in biotechnology. Match them with their exact role.

Taq polymerase is a thermostable DNA polymerase.

S1 nuclease cleaves the ds DNA at the single stranded DNA caused by the nick or a gap or some mismatch.

Restriction endonucleases are the enzymes that cut the DNA at a specific site.

Ligase is an enzyme that is used to join the nicks of the DNA.

So, the correct answer is option B.

(a) Tobacco plants are damaged severely when infested with Meloidogyne incognita. Name and explain the strategy that is adopted to stop this infestation.
(b) Name the vector used for introducing the nematode specific gene in the tobacco plant.

(a) The method adopted to develop nematode resistance in tobacco plant is RNAi (RNA Interference). It involves the following steps:

(i) Disease-causing gene was identified and isolated from the nematode Meloidogyne incognita. This gene is incorporated into the genome of tobacco plant with the help of a suitable vector.

(ii) It is introduced in tobacco plant in such a way that both sense and anti-sense mRNA is produced. These RNAs being complementary get paired to form a double-stranded RNA.

(iii) This double-stranded RNA induces RNAi in the tobacco plants which neutralize the mRNA of the nematode during actual infection. Thus nematode is unable to live such as transgenic host and therefore the tobacco plant become resistant to the pest.

(b) The vector used for introducing the nematode specific gene in tobacco plant is Agrobacterium tumefaciens.

Rearrange the following in the current sequences to accomplish an important biotechnological reaction.
(a) In vitro synthesis of copies of DNA of interest
(b) Chemically synthesized oligonucleotides
(c) Enzyme DNA-polymerase
(d) Complementary region of DNA
(e) Genomic DNA template
(f) Nucleotides provided
(g) Primers
(h) Thermostable DNA-polymerase (from Thermus aquaticus)
(i) Denaturation of ds-DNA

The correct sequence of the steps involved in biotechnological reaction is

Genomic DNA template
Denaturation of ds-DNA
Primers
Nucleotides provided
Thermostable DNA polymerase (from Thermus aquaticus)
Enzyme DNA polymerase
Chemically synthesised oligonucleotides
Complementary region of DNA
In vitro synthesis of copies of DNA of interest

Can you think and answer how a reporter enzyme can be used to monitor transformation of host cells by foreign DNA in addition to a selectable marker?

A reporter gene can be used to monitor the transformation of host cells by foreign DNA by keeping a track of its receptor gene. It act as a selectable marker to determine whether the host cell has taken up the foreign DNA or the foreign gene gets expressed in the cell.

The reporter gene and the foreign gene are placed in the same DNA construct and then inserted in the cell. Here, the reporter gene is used as a selectable marker to find out the successful uptake of foreign genes. An example of reporter genes includes lac Z gene, which encodes a green fluorescent protein in a jelly fish.

Can you suggest a method to remove oil (hydrocarbon) from seeds based on your understanding of rDNA technology and chemistry of oil?

Glycerols and fatty acids are main constituents of seed oils. Insertion of rDNA carrying the gene for any of glycerol synthesis inhibtors or fatty acid synthesis inhibitors would inhibit oil synthesis and the recombinants seeds won't have oils. Knocking out/removing the gene/genes responsible for the synthesis of oils is another option.

Explain in detail the role of restriction endonuclease in formation of recombinant DNA. Draw a labelled diagram of E.Coli cloning vector pBR 322.

A restriction enzyme (restriction endonuclease) is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are of great use in genetic engineering for restricting desired genes for incorporation. The restriction enzymes are used for the cleavage of the desired gene from the isolated DNA and the plasmid vector. There are specific recognition sites which are used for the cleavage. Certain enzymes produce sticky ends which can be ligated while the introduction of the gene in the vector.

There are various vectors which are used in genetic engineering for the introduction of the desired genes in the host cells. pBR322 is one of the common plasmid vectors. The size of this vector is 4361 bp. There is an ori site which is the site for replication. There are multiple recognition and cloning sites which are used for the insertion of genes. There are antibiotic resistance genes like ampicillin ($$amp^R$$) and tetracycline ($$tet^R$$).

What do you mean by fermentors?

The fermentor is a vessel or an apparatus which is used for the fermentation activity of the microorganisms for the commercial production of certain substances.
There are different types of fermentors depending on the type of product and the volume of the product to be produced. The common type of fermentors are tower fermenter, bubble column, air-lift, fluid bed fermenter, etc.

Write the name of the technique, by which DNA fragments can be separated.

Agarose gel electrophoresis is the technique which is used for the separation of the DNA fragments. This is the method which allows the separation of the DNA fragments on the basis of their size when there is an external supply of electric charge. The DNA is negatively charged due to phosphate backbone and thus will move towards the positive end. This method allows separation of the molecule on the basis of their size. Small fragments will be present near the positive end and large fragments will be present near the well (negative end).

Given an account of artificial chromosomes in transfer of genetic material.

The artificial chromosomes are the small extra chromosomal structures which can be used for transfer of genetic material. There are various types of artificial chromosomes which are synthesized. There are yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) which were synthesized. The human artificial chromosomes called as HAC has also been created. These are used as tools or vectors for transferring the genetic material from one organism to another. This can also be used to create genetically modified organisms which contains the desired gene responsible for the production of certain useful compounds. These can be useful for obtaining recombinant products and also be helpful in gene therapy.

Match the items in Column I with those in Column II and choose the correct answer.

Polymerase chain reaction or PCR is a technique used to amplify a single copy or a few copies of a piece of DNA to thousands of copies of a particular DNA sequence. Transformation is a transfer of genetic material from one bacterial strain to other without establishing a physical contact; bacteria take up the DNA present in the surrounding. DNA ligase is used to join DNA molecules together to form recombinant DNA. Post-translational modification of the protein is done with the help of allosteric ribozymes.

Define r-DNA technology. Give the basic steps in r-DNA technology and give any three examples of the therapeutic products produced by r-DNA technology.

Recombinant DNA (rDNA) technology is the technique of manipulating the genome of a cell or organism to change the phenotype desirably. The basic steps involved in the process are:
1. Isolating genomic DNA or a donor. The cell or organism from which the required gene is taken is called donor.
2. Fragmenting this DNA using molecular scissors (Enzymes Restriction Endonuclease).
3. Screening the fragments for the desired gene.
4. Inserting the fragments with the desired gene into a cloning vector (plasmid, cosmid or phage DNA) to develop a recombinant DNA or chimeric DNA.
5. Introducing the recombinant vector into a competent host cell.
6. Culturing these cells to obtain multiple copies or clones of the desired fragment of DNA.
7. Using these copies to Transform suitable host cells so as to express the desired gene.

Examples of the therapeutic products produced by r-DNA technology are:
1. Blood proteins: Erythropoietin
2. Human Hormones: Insulin,
3. Immune modulators : $$\alpha$$-Interferon, $$\beta$$ interferon,
4. Vaccines: Hepatitis B.

Draw a neat labelled diagram showing steps of PCR?

Answer:

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What is Recombinant D.N.A. ? Explain the process of separation and isolation of D.N.A fragment by gel electrophoresis technique.

A recombinant DNA molecule is a hybrid DNA molecule which contains segments of two or more different DNA molecules.

Agarose gel electrophoresis is mainly used to separate the DNA molecule on the agarose gel matrix. The negatively charged molecules of DNA are loaded as a sample on the deeply carved wells on the gel. The loaded sample is induced by a strong electric field which leads to the separation of DNA on the basis of mass and the charge on the DNA. As all the DNA molecules have the same charge so they get aligned on the basis of mass. The smaller DNA molecules move faster and get aligned at the top with the larger segment at the bottom. DNA extraction from agarose gel is divided into many steps. A DNA fragment that is isolated from a cell's nucleus by treating the cells with multienzymes in proper conditions. Once DNA is extracted, to confirm it, it is run on agarose gel. One can see DNA bands in agarose gel by autoradiography. Agarose gel containing extracted DNA shows dark bands when exposed to UV light when the DNA content is high and shows light bands when the DNA content is less. This DNA can be extracted and purified from the gel by the process of elution. DNA is passed through silica columns. The DNA has an affinity for silica gets attached to the membrane and rest of the contents pass through the column.

Write the steps in specific sequence of recombinant DNA technology. Describe the method of isolation of the genetic material (DNA).

Genetic engineering is also called genetic modification. This method involves the manipulation of the genome of any organism using biotechnology techniques.

The gene may be inserted into the host genome. The gene is first isolated from the cell. The desired gene is restricted with the help of specific restriction enzymes. These enzymes are also used to cleave the plasmid vector. The desired gene is inserted in the plasmid vector with the help of enzyme ligase. This results in the formation of recombinant DNA. Many different segments of DNA can be attached in the vector. The plasmids are cloned in the bacterial cells by using molecular cloning methods.

There are various different methods which can be used for the isolation of the genetic material. The common method which is used includes the alkali or the acid treatment which results in the breakdown of the cell wall. The parts of the cell are separated by using differential centrifugation. This is the type of separation of the different organelles on the basis of density. The DNA can be separated, PCR can be performed and then isolated by using gel electrophoresis.

Explain briefly the various processes of recombinant DNA technology.

DNA recombinant technology is a technique where the selected DNA of one organism is introduced to combine with the DNA of another organism acquires the genetic abilities of the donor.

The basic steps involved in the process of DNA technology are as follows.

(i) The genomic DNA is isolated from a donor.

(ii) Using restriction enzymes such as endonucleases the DNA is fragmented. Endonuclease enzymes are known as molecular scissors as they produce nick in the DNA fragment at the desired place.

(iii) The fragments were taken out are then screened for the desired gene.

(iv) Fragments of DNA with the desired gene are inserted into a cloning vector. Cloning vector can be a plasmid, cosmid or even a phage DNA. This insertion makes recombinant DNA or DNA (also known as chimeric DNA).

(v) The recombinant vector is introduced into a competent host cell.

(vi) These cells are later cultured to obtain multiple copies or clones of the desired fragment of DNA.

(vii) These copies are used to transform suitable host cells which express the desired gene.

Name the three important steps involved in PCR process.

Denaturation: The double-stranded DNA is heated up to 98$$^o$$C which causes the hydrogen bonds to break and the two strands get separated.
Annealing: The two sets of primers are added which bind to the appropriate complementary segment of DNA strand.
Extension: The Taq polymerase enzyme polymerizes the nucleotide chain using the nucleotides provided in the medium and by using the template strand.

Mention the different steps of process of recombinant DNA technology.

DNA recombinant technology is a technique where the selected DNA of one organism is introduced to combine with the DNA of another organism acquires the genetic abilities of the donor.

The basic steps involved in the process of DNA technology are as follows.

(i) The genomic DNA is isolated from a donor.

(ii) Using restriction enzymes such as endonucleases the DNA is fragmented. Endonuclease enzymes are known as molecular scissors as they produce nick in the DNA fragment at the desired place.

(iii) The fragments were taken out are then screened for the desired gene.

(v) The recombinant vector is introduced into a competent host cell.

(vi) These cells are later cultured to obtain multiple copies or clones of the desired fragment of DNA.

(vii) These copies are used to transform suitable host cells which express the desired gene.

Write the names of two antibiotic resistant genes found in plasmid $$P^{BR322}$$.

pBR322 is a plasmid vector in which p stands for plasmid and BR stands for Bolivar and Rodriguez, the scientists who constructed it. The plasmid pBR322 vector carries the genes for tetracycline ($$tet^R$$) and ampicillin ($$amp^R$$) resistance. These genes are useful to identify and select the transformants and non-transformants.

What is the full form of PCR? How is it useful in biotechnology?

The term PCR stands for polymerase chain reaction. It is a technique which is used in molecular biology to amplify a single copy of DNA to obtain millions of copies. The gene which is used for insertion in the vectors can be amplified by using this technique. This technique is commonly used in DNA fingerprinting method for the amplification of the DNA sample which is obtained from the site of investigation. There can be DNA samples obtained and amplify for detection in the paternity testing method. This method helps in the diagnosis of mutation which takes place in genetic disorders. Any gene can be amplified and studied by using this technique. For genetic experiments, the amount of sample which is required is more and so this technique is useful. The DNA obtained from the organism will be less which can be amplified and used for the experiments.

Match the columns and choose the right option.

A-3,B-4,C-2,D-1

Solution : Bacillus thuringiensis produces a insecticidal protein called cry toxin. Agrobacterium tumefaciens produces Ti plasmid used in gene transfer. Thermus acquaticus is a microbe from which we get Taq polymerase used in PCR as a thermostable DNA polymerase.E.coli ha restriction endonuclease that helps in cutting double stranded DNA.

So the correct answer is " A-3,B-4,C-2,D-1"

How is the amplification of gene done using the technique of PCR? Explain with the help of diagram.

The amplification of gene is done by using the technique of PCR. PCR stands for Polymerase Chain reaction. PCR enables the production or amplification of billions of copies of an original piece of DNA in the tube with minutes or hours. A single PCR reaction involves three temperature-dependent steps, described as follow:

Denaturation: The starting solution is heated; usually to 94 degree Celsius. The high temperature breaks the hydrogen bonds between the two strands to the original DNA double helix, providing the necessary single-standard templates.
Annealing: After just a couple of minutes at that temperature, the reaction mixture is quickly cooled, usually to somewhere between 50 degree Celsius and 60 degree Celsius. It is then held for less than a minutes at this lower temperature- which is enough time for the primers to bind to their complementary sequences on the single-standard templates.
Primer extension (polymerization): The sample is next heated to 72 degree Celsius for some time, during which time the DNA polymerase adds nucleotides to the primer, synthesizing a new DNA strand using only the template sequences that bind the primers.

If the process of DNA replication is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made at the end of 30 PCR cycles. It is possible to generate $$2^n$$ molecules after 'n' number of cycles.

What does PCR stand for? Describe the different steps of PCR.

PCR stands for polymerase chain reaction. It is a process which is used to create copies of DNA fragment. In this, PCR reaction mixture contains Taq polymerase, primers, template DNA and nucleotides There are three steps of PCR namely, denaturation step, annealing step and extension step.

1. Denaturation step: In this step, the reaction is heated at 94$$^0$$C in order to denature the DNA strands. This results in a single-stranded template.

2. Annealing step: In this step, the reaction is cooled down to 55$$^0$$C to 65$$^0$$C in order to bind primer to their complementary sequences on the single-stranded template DNA.

3. Extension: This is the last step of PCR process. In this step, the temperature of reaction is raised to 72$$^0$$C so that Taq polymerase enzyme synthesizes new strands of DNA by extending primer.

This cycle repeats 25 to 35 times in the entire process.

Match the columns and choose the right option.

ELISA involves antigen-antibody interaction followed by chemiluminescence. It is used in the early diagnosis of disease.
PCR (Polymerase chain reaction) involves the amplification of the DNA.
Gold-coated DNA is used as a microprojectile in biolistics or gene gun methods. This method is suitable for the transformation of plants.
Micro-injection is the direct introduction of DNA into the nucleus of animal or plant cells.

So, the correct answer is "A-1, B-2, C-4, D-3".

Explain the role of restriction endonucleases in recombinant DNA technology. Name the endonuclease that was first discovered.

Restriction endonucleases are enzymes that cut double-stranded DNA at very specific recognition sites. They were originally discovered in bacteria that use them to restrict the growth of viruses but are now among the workhorse enzymes of biotechnology and recombinant DNA research. The endonuclease that was first discovered was Hind II.

Mention the role of molecular scissors in recombinant DNA technology.

Restriction endonucleases are also called as molecular scissors.
Restriction endonucleases make random cuts in DNA, but the restriction endonucleases are site-specific.
Some examples of the restriction endonucleases are Bam HI, Eco RI, Bgl II, Hae II, Hind II.

Give an account of the Blue-White Method of selection of recombinants.

Recombinant DNA technology involves several steps in specific sequence such as isolation of DNA, fragmentation of DNA by restriction endonucleases, isolation of a desired DNA fragment, ligation of the DNA fragment into a vector, transferring the recombinant DNA into the host, culturing the host cells in a medium at large scale and extraction of the desired product. The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in such experiments. Recombinant DNA is inserted into a competent host cell viable for transformation, which are then grown in presence of X – gal. Cell transformed with vectors containing Recombinant DNA will produce white colonies, cells transformed with non – recombinant plasmids (i.e. only the vector) grow into blue colonies. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used.

How and why is bacterium Thermus aquaticus employed in recombinant DNA technology? Explain.

Bacterium Thermus aquaticus is a heat labile bacterium which is obtained from hot springs of temperatures more than 97-degree centigrade. This bacterium produces an enzyme Taq polymerase which is thermostable and is active even at high temperature also and results in the denaturation of double-stranded DNA. These enzymes are widely used in the recombinant DNA technology mainly PCR (Polymerase chain reaction). In PCR these Taq polymerase enzymes help in the amplification of desired DNA to obtain better recombinants.

Mention the three steps involved in each cycle of Polymerase-chain Reaction (PCR). How is repeated amplification of DNA made possible during PCR

In a PCR reaction, DNA polymerase synthesises a new DNA strand complementary to the DNA template by adding nucleotide to the primers. The above components are mixed together in PCR tubes and run on a thermal cycler, where they go through multiple cycles involving the following reactions:

Denaturation – This step involves heating the reaction mixture to a high temperature (94°C). This denatures the double-stranded DNA template into single-strands by breaking the weak hydrogen bonds between the two DNA strands.

Annealing – In this step, the reaction mixture is brought to a temperature of 54-60°C, to allow the primers to bind (anneal) to their complementary sequence in the template DNA.
Elongation/Extension – In this step, the DNA polymerase sequentially adds nucleotides to the primers and extends it in the 5′ to 3′ direction.

One cycle of denaturation, annealing, and elongation amplifies the double-stranded DNA template to give two pieces of double-stranded DNA. The cycles keep repeating to produce more and more copies, increasing the number of copies exponentially.

Image result for Mention the three steps involved in -each cycle of Polymerase-chain Reaction (PCR). How is repeated amplification of DNA made possible during PCR

What is recombinant DNA technology called popularly ? Name the vectors and enzymes used in this technique.

The DNA recombinant technology is also known as genetic engineering. It deals with the alteration of the genetic makeup of cells by deliberate and artificial means. The various vectors used in this technique are plasmid vector, bacteriophage vector, cosmid vector, BACs ( bacterial artificial chromosomes ), YACs ( yeast artificial chromosomes ), MACs ( mammalian artificial chromosomes ). Some of the enzymes used in genetic engineering are BAM HI, Bgl II, Eco RI, Hae II and Hind III.

Name and explain the techniques used in the separation and isolation of DNA fragments to be used in recombinant DNA technology.

The technique used for separation of DNA fragments is called gel electrophoresis. The technique is based on the principle that - when a charged molecule is placed in an electric field they move towards the positive or negative side according to their charge. When the electric field is applied in DNA fragments being negatively charged,move through the gel towards the anode and gets separated.

Explain the contribution of Thermus aquaticus in the amplification of a gene of interest.

Thermostable DNA polymerase enzyme is extracted from Thermus aquaticus. Polymerase chain reaction is the process of making multiple copies of the DNA segment in vitro. The double stranded is denatured by using high temperature. DNA polymerase is used to add nucleotides on the DNA strand. High temperature inactivate the DNA polymerase. Taq polymerase can withstand the high temperature used for the denaturation and separation of DNA.

(a) Why are restriction endonucleases so called ? (b) What is a palindromic nucleotide sequence ? How do restriction endonucleases act on palindromic sites to create sticky ends ?

1. Restriction enzymes are DNA-cutting enzymes found in bacteria. In order to be able to sequence DNA, it is first necessary to cut it into smaller fragments, because they cut within the molecule, they are often called restriction endonucleases.

2. A palindromic sequence is a nucleic acid sequence on double-stranded DNA or RNA wherein reading 5' (five-prime) to 3' (three prime) forward on one strand matches the sequence reading 5' to 3' on the complementary strand with which it forms a double helix.

3. Restriction enzymes recognize a specific DNA sequence, normally between four and eight base pairs long. The A A T T C sequence to the 3' direction of the G is called an overhang. The restriction enzyme Eco RI makes sticky ends when it cuts DNA. If both sequences are cut with Eco RI, they can be joined together.

(a) With the help of diagrams show the different steps in the formation of
recombinant DNA by action of restriction endonuclease enzyme Eco RI (b) Name the technique that is used for separating the fragments of DNA cut by restriction endonucleases.

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2. Gel electrophoresis.

DNA are negatively charged, forced to move towards anode, electric field agarose gel matrix, separate according to their size / sieving effect, smaller fragments moves faster and further than the larger. Since DNA fragments are negatively charged molecules. They can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. The most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate according to their size through sieving effect provided by the agarose gel.Hence, the smaller the fragment size,the farther it moves.

(a)EcoRI is a restriction endonuclease. How is it named so? Explain (b) Write the sequence of DNA bases that the enzyme recognises. Mention the point at which the enzyme makes a cut in the DNA segment.

1. Eco RI is named after the enzyme found in E.coli RY13.

2. 5' G^AATTC 3'

3' CTTAA^G 5'

Any recombinant DNA with a desired gene is required in billion copies for commercial use. How is the amplification done? Explain.

1. Amplification refers to the process of making multiple copies of the DNA segment in vitro.

2. It employs polymerase chain reaction (PCR).

3. The process was designed by K.Mullis.

4. This technique involves three main steps :

Denaturation
Primer annealing and
Extension of primers.

5. The double-stranded DNA is denatured by using high temperature.

6. Two sets of primers are used.Primers are the chemically synthesized short segments of DNA that are complementary to the segment of DNA.

7. DNA polymerase is the enzyme used to make copies of DNA by using the genomic template DNA and the primer.

(a) Mention the number of primers required in each cycle of polymerase chain
reaction (PCR). Write the role of primers and DNA polymerase in PCR., (b) Give the characteristic feature and source organism of the DNA polymerase in PCR.

Two sets of primers and the enzyme DNA polymerase are required in each cycle of polymerase chain reaction.
Role of primers and DNA polymerase : Primers are necessary to start the functioning of DNA polymerase. DNA polymerase extends the primers using the nucleotides provided in the reaction and the genomic DNA as the template. The segment of DNA can be amplified to approximately billion times. Such repeated amplification is achieved by the use of a thermostable is achieved by the use of a thermostable DNA polymerase, which remains active during the high-temperature induced denaturation of double stranded DNA.
DNA polymerase used in PCR reaction is known as Taq polymerase, which is obtained from a bacterium Thermus aquaticus. It remains active during the high-temperature induced denaturation of double-stranded DNA.

(a) Illustrate the recognition sequence of Eco RI and mention what such sequences are called (b) How does restriction endonuclease act on a DNA molecule?

1. 5' G^AATTC 3'

3'CTTAA^G 5'

These sequences are called as palindromic sequences.

2. When a restriction endonuclease recognises a sequence, it snips through the DNA molecule by catalysing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides.

Describe the process of gene amplification for rDNA technology experiments.

Amplification of a gene sample of interest is carried out using PCR in the following three steps:

1. Denaturation-

•The double-stranded DNA is denatured by applying a high temperature of about 95 degree Celsius for 15 seconds.

•Each separated single-stranded strand now acts as a template for the synthesis of a new DNA strand.

2. Annealing-

•Two sets of primers are added which anneal at the 3’ end of each separated strand. They mark the beginning of replication.

•It occurs at about 40 to 60 degree Celsius depending on the length and sequence of primers.

3. Extension

•A thermostable DNA polymerase, Taq polymerase extends the primers by adding nucleotides complementary to the template provided in the reaction.

•Polymerisation is usually carried out at 72 degree Celsius.

Why is the enzyme cellulase used for isolating genetic material from plant cells but not for animal cells?

Plant cells have cellulose in their cell wall which can be degraded using the enzyme cellulase for isolating the genetic material DNA. On the other hand, animal cell wall does not contain cellulose. Hence, cellulase is not required for isolating DNA.

How is the action of exonuclease different from that of endonuclease?

Exonuclease are enzymes that cleave the nucleotides at the end of the DNA molecule.The cleavage by exonuclease results in the formation of nucleotides or nucleosides.

For example- BAL31

Endonuclease are enzymes that cleave the bonds of the DNA from within the molecule(middle part) at specific site. The cleavage by endonuclease results in the formation of oligonucleotides(Four nucleotide residues).For example- S1 nuclease, DNAses, Restriction endonucleases.

Eco RI is used to cut a segment of foreign DNA and that of a vector DNA to form a recombinant DNA. Show with the help of schematic diagrams.(i)
The set of palindromic nucleotide sequence of base pairs the Eco RI-will
recognise in both the DNA segments. mark the site at which Eco RI will act and-cut both the segments., (ii) Sticky ends formed on both the segments where the two DNA segments will join later to forma recombinant DNA.

2. Palindromic nucleotide sequence of EcoR I is

5' GAATTC 3'

3' CTTAAG 5'

3. Each restriction endonuclease functions by 'inspecting' the length of a DNA sequence. Once it finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of the double helix at specific points in their sugar-phosphate backbones. Each restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA. Restriction enzymes cut the stand of DNA between the same two bases on the opposite strands. There are overhanging stretches called sticky-ends on each strand. They form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

How are the following used in biotechnology ?
(a) plasmid DNA (b) recognition sequence (c) gel electrophoresis

1. Plasmid DNA : The construction of the first recombinant DNA molecule emerged from the possibility of linking a gene encoding antibiotic resistance with a native plasmid of Salmonella typhimurium. Stannely Cohen and Herbert Boyer accomplished this in 1972 by isolating the antibiotic resistance gene by cutting out a piece of DNA from a plasmid that was responsible for conferring antibiotic resistance.

2. Recognition sequence : The first restriction endonuclease Hind II, whose functioning depended on a specific DNA nucleotide sequence, was isolated and characterised five years late.This specific base sequence is knows as the recognition sequence for Hind II.

3. Gel electrophoresis : The cutting of DNA by restriction endonuclease results in the fragments of DNA. These fragments can be separated by a technique knows as gel electrophoresis.

Match column I with column II with respect to the nomenclature of restriction enzyme EcoRI and select the correct answer from the given codes.

EcoRI is the restriction endonuclease enzyme in which E stands for name of genus , Co stands for name of species , R stands for the name of strain from which it is isolated and I stands for its identification of number i.e; it is the first strain of particular species.

So, the correct option is 'Option A-2 , B-3 , C-4 , D-1' .

List the key tools used in recombinant DNA technology.

The key tools used in recombinant DNA technology are

1.Isolation of DNA

2.Fragmentation of DNA by restriction endonucleases

3.Isolation of the desired DNA fragment.

4.Amplification of the gene of interest.

5.Ligation of the DNA fragment into a vector using DNA ligase.

6.Transfer of recombination DNA into the host and many more.

Name the source of the DNA polymerase used in PCR technique. Mention why it is used.

T.aquaticus or Thermus aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions like high temperature required during PCR. Therefore is the source of the DNA polymerase used in PCR technique.

Explain briefly PCR.

PCR stands for polymerase chain reaction. In this reaction, multiple copies of the gene of interest are synthesized in vitro under three steps.
(i) Denaturation : In this, double stranded DNA is converted to the single stranded often achieved by heating or alkaline conditions. This is called "melting" of DNA.
(ii) Annealing : The two sets of primers (small chemical synthesized oligonucleotides that are complementary to the regions of DNA) undergo biochemical process of annealing at an optimum temperature of $$40-65^0$$C.
Extension: The enzyme DNA polymerase extends the primers using the nucleotides provided in the reaction and the genomic DNA as template.
In the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times. Such repeated amplification is achieved by the use of a thermostable DNA polymerase and the amplified fragment if desired can be used to ligate with a vector for further cloning.

Expand and mention one application of PCR.

The polymerase chain reaction is used to reproduce selected sections of DNA or RNA for analysis. Previously, amplification of DNA involved cloning the segments of interest into vectors for expression in bacteria. The PCR has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, microarrays, forensics, and paternity testing.

Match the scientists in column I with their related discoveries in column II and select the correct option from the given codes.

The father of genetic engineering is Paul Berg . He creates the recombinant DNA technology in 1972.
Kary Mullis invents the polymerase chain reaction in 1993.
Arber, Smith and Nathan discovered the restriction endonuclease enzymes in 1978.
Stanley Cohen and Herbert Boyer constructed artificial recombinant DNA for the first time. They got the idea of linking a gene encoding for antibiotic resistance with a native plasmid of Salmonella typhinurium.

So, the correct answer is ' A-3, B-1, C-4, D-2' .

Write any four ways used to introduce a desired DNA segment into a bacterial cell in recombinant technology experiments.

The four ways used to introduce the desired DNA segment into a bacterial cell in recombinant technology experiments are

1.Microinjection

2.Heat shocking method

3.Gene-gun or biolistics method.

4.Using disarmed pathogen vectors.

Describe the characteristics a cloning vector must possess.

The main characteristics of a cloning vector is

It needs to have a unique restriction sites.If it posses to many restriction sites then it will break into several pieces.

It needs to be a DNA molecule so it can be cloned with gene.

There must be a presence of origin of replication in sequence where the replication starts.

The vector must be able to replicate into the host system.

Any recombinant DNA with a desired gene is required in billion copies for commercial use. How is the amplification done? Explain.

Amplification refers to the process of making multiple copies of the DNA segment in vitro.

1. It employs polymerase chain reaction (PCR).

2. The process was designed by K.Mullis.

3. This technique involves three steps.

Denaturation, annealing, and extension.

4. The double-stranded DNA is denatured by using high temperature.

5. Two sets of primers are used. Primers are the chemically synthesized short segments of DNA that are complementary to the segment of DNA.

6.DNA polymerase is the enzyme used to make copies of DNA by using the genomic template DNA and the primer.

Identify $$A$$ and $$B$$ illustrations in the following

The given structure A shows a palindrome which cuts the DNA from the specific site after recognizing a particular sequence of DNA. Here is the sequence as 5'-CAATTC-3'.

In the second image, the B portion represents the desired part of the gene which is being added to the plasma for its transfer into the host cells DNA. This is generally attached to the origin of replication site from where the replication is easy.

Explain the role of molecular scissors in recombinant DNA technology.

Restriction enzymes provide protection to bacteria against bacteriophages. They are used in genetic engineering as they cut DNA at a specific sequence and form recombinant molecules.

Explain the role of Ti plasmids in biotechnology.

Ti plasmid (Tumor-inducing plasmid) refers to the plasmid of Agrobacterium tumefaciens. A.tumefaciens is a plant pathogen and is known to produce tumors in the plant it infects. Role of Ti plasmid in biotechnology is that Ti plasmid can be modified into a cloning vector by removing the genes responsible for pathogenicity.

What does PCR stand for? What is the principle of PCR? Name the different steps in PCR reaction.

PCR stands for Polymerase chain reaction. Polymerase chain reaction (PCR) is a technique used to make numerous copies of a specific segment of DNA quickly and accurately. The polymerase chain reaction enables investigators to obtain large quantities of DNA that are required for various experiments and procedures in molecular biology, forensic analysis, evolutionary biology, and medical diagnostics.

The steps in PCR reaction are,

1.Denaturation of the template into single strands

2.Annealing of primers to each original strand for new strand synthesis

3.Extension of new DNA strands from the primers.

How does restriction endonuclease act on a DNA molecule?

Recognition sequences are randomly distributed throughout the DNA. When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by catalyzing the hydrolysis of the bond between adjacent nucleotides.

With the help of diagrams show the different steps in the formation of recombinant DNA by action of restriction endonuclease enzymes EcoR$$1$$.

Name the selectable markers in the cloning vector $$pBR 322$$? Mention the role they play.

In the cloning vector pBR322, ampicillin and tetracycline resistance genes are the selectable markers. The role they play is that they help in the selection of transformed cells from non transformed cells. They also help distinguish recombinant cells from non-recombinant cells.

Name the technique that is used for separating the fragments of DNA cut by restriction endonucleases.

The technique is called Gel Electrophoresis. DNA is negatively charged, forced to move towards the anode, electric field agarose gel matrix, separated according to their size/sieving effect, smaller fragments move faster and further than the larger. They can be separated by forcing them to move towards the anode under an electric field through a medium/matrix.

How is the separated DNA visualised and extracted for use in recombinant technology ?

The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation. We can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light.

The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

How are the DNA fragments separated by gel electrophoresis is visualized and separated for use in constructing recombinant DNA?

DNA molecule is negatively charged, it can be pulled through the gel by an electric field. Small DNA molecules move more quickly through the gel than larger DNA molecules. As they are negatively charged they move towards the anode under an electric field. Then the separated bonds cut from the gel and get extracted by using a conventional technique. The eluted DNA fragments are then purified and used in recombinant DNA.

Write the type of matrix used in the technique for the separation of DNA fragments.

DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field through a medium/matrix. Nowadays the most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. Hence, the smaller the fragment size, the farther it moves.

What would happen when one grows a recombinant bacterium in a bioreactor but forget to add antibiotic to the medium in which the recombinant is growing?

Presence of antibiotics in the medium where recombinant bacteria are growing makes sure that the bacteria retain the plasmid containing the antibiotic resistant gene (as well as the gene of interest). In the absence of antibiotics, the pressure will be eliminated and the bacteria will not maintain the copy number of the plasmid. Hence, the plasmid will be lost (so as the gene of interest). So, in such a bioreactor the quantity of the desired product will be greatly reduced.

While cloning vectors, which of the two will be performed by biotechnologists bacteriophages or plasmids. Justify with reason.

Plasmids used as vectors maintain a modified origin of replication that allows their replication within the host, and they contain a gene for antibiotic resistance which ensures that, following treatment with a high dose of antibiotic, all viable bacterial colonies will contain several copies of the plasmid.

How does the wells gets charged in the Gel electrophoresis?

Gel electrophoresis is a technique used to separate DNA fragments according to their size. DNA samples are loaded into wells (indentations) at one end of a gel, and an electric current is applied to pull them through the gel. When the electric field is applied to the set up the DNA being negatively charged they move towards the positive electrode. The DNA moves in the according to there size as the DNA with molecular weight move slowly while the DNA with lightweight move faster. By this movement, the bands are created which we can observe in the UV illuminator.

What are vectors?

Vector is an organism that does not cause disease itself but which spreads infection by conveying pathogens from one host to another. Species of mosquito, for example, serve as vectors for the deadly disease Malaria.

Restriction enzymes that are used in the construction of recombinant DNA are endonucleases which cut the DNA at 'specific-recognition sequence'. What would be the disadvantage if they do not cut the DNA at specific-recognition sequence?

The restriction endonucleases cleave the DNA molecules at the specific sites called the restriction sites, that are made of palindromic sequences. If the sequence-specific cuts are not made by the restriction endonucleases, it would become impossible to target the specific gene. The random cuts would result in variable lengths of DNA fragments and the joining of the target gene with vector DNA would have become cumbersome as restriction endonuclease create either the sticky ends or the blunt ends. In both the cases, it is easy to monitor the ligation process. In other words, the r-DNA technology would not have gained the momentum.

Mention four features of $$pBR_{322}$$.

Bacteria have several copies of circular DNA in addition to a chromosome. They are called plasmids. They contain genes and replicate independently. Hence they are carried from generation to generation. pBR$$_3$$$$_2$$$$_2$$ is one of the earliest genetically engineered plasmid. It contains the genes for resistance to ampicillin and tetracycline and can be amplified with PCR. The molecular weight is 2.83 x 106 daltons.
1073777_1170749_ans_65e51b3aab2648379b34b3440c4d74d0.PNG

1073777_1170749_ans_65e51b3aab2648379b34b3440c4d74d0.PNG

Define Insertional inactivation.

Insertional inactivation technique of recombinant DNA technology used to select bacteria that carry recombinant plasmids; a fragment of foreign DNA is inserted into a restriction site within a gene for antibiotic resistance, thus causing that gene to become nonfunctional. It is often used to identify recombinant vectors in gene cloning and in turn to distinguish a recombinant vector from a non-recombinant vector. For example, insertion of a piece of foreign DNA into a cloning site which is located on an antibiotic-resistant gene on the vector can lead to loss of the antibiotic resistance phenotype by insertional inactivation. The recombinant vector will, therefore, specify antibiotic sensitivity, whilst the non-recombinant vector will specify antibiotic resistance.

a) Why have DNA fragments in band 'D' moved farther away in comparison to those in band 'C'
b) Identify the anode end in the diagram.
c) What does E represent in the above diagram?

Given here is a diagram for the technique of gel electrophoresis in which the band of DNA is separated on the basis of the size of the strands.

The DNA segments which are thinner move forward as they can pass through the sieves of the agar-agar Matrix.

As we can see in the diagram there are four different bands.

The DNA is negatively charged so it moves towards the positively charged anode, therefore, the anode and is the right side of the diagram i.e B end.

In the Bands, the DNA fragments move forward because they are thinner in size as compared to the bands above them, and they can move through the matrix of the agar-agar as they are thin in size.

E represents the standard band.

What are restriction enzymes? Mention any two restriction enzymes and their source.

A restriction enzyme is an enzyme that cleaves DNA into fragments at or near specific recognition sites within the molecule known as restriction sites. Restrictions enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses.

EcoRI: EcoRI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species E. coli. The Eco part of the enzyme's name originates from the species from which it was isolated, while the R represents the particular strain, in this case, RY13. The last part of its name, the I, denotes that it was the first enzyme isolated from this strain. EcoRI is a restriction enzyme that cleaves DNA double helices into fragments at specific sites.
BamH I: BamH I isolated from Bacillus amyloliquefaciens is a type II restriction endonuclease, having the capacity for recognizing short sequences (6 b.p.) of DNA and specifically cleaving them at a target site. BamH I binds at the recognition sequence 5'-GGATCC-3' and cleaves these sequences just after the 5'-guanine on each strand. This cleavage results in sticky ends which are 4 b.p. long. In its unbound form, BamH I displays a central b sheet, which resides in between α-helices.

Given is the diagram of agarose gel kept under UV light.
Mention the positive and negative terminals.

Gel electrophoresis is a technique used to separate DNA fragments according to their size.
DNA samples are loaded into wells (indentations) at negative end of a gel, and an electric current is applied to pull them through the gel.
DNA fragments are negatively charged, so they move towards the positive electrode. Because all DNA fragments have the same amount of charge per mass, small fragments move through the gel faster than large ones.
Hence, A is the negative terminal while B represents positive terminal.

a) How do DNA fragments migrate and resolve in a Gel electrophoresis?
b) How lane one is different from lane 2,3 and 4 in the Gel electrophoresis set up?
c) How pure DNA fragments are made observable in the visible light?

The DNA fragments in the wells of gel electrophoresis migrate under the influence of current.
Lane 1 is different than 2, 3 and 4 as there is no migration of DNA fragments.
Physical properties like staining, fluorescence, UV absorption, radioactivity etc are used to observe DNA fragments in visible light.

What modification is done on the Ti plasmid of Agrobacterium tumefaciens to convert it into a cloning vector?

Ti plasmid in Agrobacterium has the ability to induce tumor in plants. This plasmid is 'disarmed' by suitable modification and then it can be used as a cloning vector for delivering gene of interest to plants and animals.

What is DNA probe?

DNA probes are oligonucleotides used to detect the complementary sequences. DNA probes highlight the sequence that has been separated by gel electrophoresis. Target sequence bind with probe can be seen under UV light.

Would you choose an exonuclease while producing a recombinant DNA molecule?

Exonuclease removes nucleotides from the ends of the DNA and hence it cannot help in isolating specific segments of DNA using restriction enzymes, endonucleases . So, exonuclease cannot be used for making a recombinant DNA molecule.

How is copy number of the plasmid vector related to yield of recombinant protein?

A higher number of copies of plasmid vector helps in producing a large quantity of recombinant protein.

A recombinant DNA molecule was created by ligating a gene to a plasmid vector. By mistake, an exonuclease was added to the tube containing the recombinant DNA. How does this affect the next step in the experiment i.e. bacterial transformation?

When a DNA molecule is created by ligating a gene to a plasmid vector, It becomes a circular DNA which is ready to replicate in host organism. After this stage, addition of exonuclease is not going to affect the process because the DNA does not have a free end and hence enzyme exonuclease will not get a substrate to show its action. So, in this experiment; bacterial transformation is not going to be disturbed.

Write the types of restriction enzyme. "Synthesis of recominant DNA molecule is possible only when the vectors and source DNA is cut by the same restriction enzyme" explain reason.

Restriction endonucleases are a class of enzyme that cut DNA molecules. Restriction enzymes belong to a larger class of enzymes called nucleases. These are of two kinds:
I. Exonucleases remove nucleotides from the ends of the DNA.
II. Endonucleases make cuts at specific positions within the DNA.
There are four classes of restriction endonucleases:
Types I, II,III and IV.

All types of enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates. They differ in their recognition sequence, sub unit composition, cleavage position, and cofactor requirements.

"Synthesis of recombinant DNA molecule is possible only when the vector and source DNA is cut by the same restriction enzyme"

Explanation:
Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded portions at the ends. Them are overhanging stretches called sticky ends on each strand. These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase. Restriction endonucleases are used in genetic engineering to form 'recombinant' molecules of DNA, which are composed of DNA from different sources/genomes. When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of 'sticky-ends' and, these can be joined together (end-to-end) using DNA ligases. Normally, unless one cuts the vector and the source DNA with the same restriction enzyme, the recombinant vector molecule cannot be created.

A person wanted to isolate DNA from cells of a human to perform his experiment on recombinant DNA technology. He used RBC cells and WBC cells for the same and loaded well 1 (sample from RBC) and well 2 (sample from WBC). After performing gel electrophoresis he found the following patterns of fragments in the gel. Why do you think Well 1 has no fragments? Note: No DNase was used.

RBC cells do not have a nucleus hence they do not have DNA. This the reason why no DNA fragments are there from well 1.

A plasmid DNA and a linear DNA (both are of the same size) have one site for a restriction endonuclease. When cut and separated on agarose gel electrophoresis, plasmid shows one DNA band while linear DNA shows two fragments. Explain.

When treated with a restriction endonuclease circular DNA opens up to resemble a single linear DNA. A Linear DNA is divided into two fragments after cleavage. Hence, circular DNA shows one band, while linear DNA shows two bands when run on agarose gel electrophoresis.

DNA fragments	$$A$$	$$B$$	$$C$$
Size (in Base Pairs)	$$700$$	$$1500$$	$$3000$$

Biotechnology: Principles And Processes - Class 12 Medical Biology - Extra Questions

Answer in 3 to 5 sentences:Draw a neat labelled diagram of a typical agarose gel electrophoresis.

Manipulating with nucleic acid is a trend in biotechnology.(a) Name any one organism used as vector.(b) What are DNA polymerase ?

How will you transfer a whole chromosome into a fertilized egg?

Name the bacterium that yields a thermostable DNA polymerase.

What is main function of gel electrophoresis?

What is particle gun?

What chemical is used in vectorless gene transfer?

Which cloning vector was discovered for the first time?

Name an eukaryotic organism that has plasmids, and can be used as a host in gene cloning experiments.

What was the first recombinant DNA based product, produced and marketed in India?

What is called a 'probe'?

Name the compound used for staining the isolated DNA in the gel.

(a) Why must a cell be made 'competent' in biotechnology experiments? How does calcium ion help in doing so?(b) State the role of biolistic gun in biotechnology experiments.

How is the amplification of a gene sample of interest carried out using Polymerase Chain Reaction (PCR)?

Name the source organism that possesses Taq polymerase. What is so special about the function of this enzyme?

How does a restriction nucleases function? Explain.

Why is the enzyme cellulase needed for isolating genetic material from plant cells and not from the animal cells?

Expand the following and mention one application of each:(i) PCR (ii) ELISA

How are 'sticky ends' formed on a DNA strand? Why are they so called?

Explain with the help of a suitable example the naming of a restriction endonuclease.

Mention the type of host cells suitable for the gene guns to introduce an alien DNA.

Name and describe the technique that helps in separating the DNA fragments formed by the use of restriction endonuclease.

State the role of DNA ligase in biotechnology.

Draw a schematic sketch of pBR 322 plasmid and label the following in it.(a) Any two restriction sites(b) Ori and rop genes(c) An antibiotic resistant gene

Make a chart(with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces.

Do eukaryotic cells have restriction endonucleases? Justify your answer.

Explain briefy.(a) PCR(b) Restriction enzymes and DNA(c) Chitinase

Explain the use of Agrobacterium tumifaciens in gene cloning.

Give a brief account of the tools of recombinant DNA technology.

What is the name of the sequence of base pairs which reads same on the two strands of DNA when orientation of reading is kept same?

What is downstream processing?

How to produce viruses as effective vectors?

What is palindrome in DNA?

How does one visualize DNA on an agar gel?

With the help of a neat and labelled diagram describe steps in recombinant DNA technology.

Explain the significance of 'palindromic nucleotide sequence' in the formation of recombinant DNA.

Expand 'YAC' and mention what was it used for.

Write the use of restriction endonuclease in the above process.

Figure representing the reactions associated with Polymer Chain Reaction (PCR).Name the steps A, B, C in the progress.

Answer in 40 to 80 words:Explain in brief the separation and isolation of DNA fragments.

Given an example of the source of thermostable enzyme DNA polymerase.

Use of a thermostable DNA polymerase from the bacterium, Thermus aquatics, made it possible to generate a billion copies of DNA in a very short time using a process. Name the process.

Define polymerase chain reaction.

Match the lists and find the correct option.

Name two commonly used vectors in genetic engineering.

Describe the steps of PCR technique.

Enlist the basic steps involved in recombinant DNA technology.

List the key tools used in recombinant DNA technology.

Differentiate between-: Gene cloning and cell cloning

Very short answer type.Selfing is a kind of inbreeding.

What are molecular scissors ? Give one example. Explain their role in recombinant DNA technology.

Do you know what palindromes are?

Write the methods to introduce foreign DNA into the host cells.

Draw a neat labelled diagram of PBR322.

"A very small sample of tissue or even a drop of blood can help determine paternity." Provide scientific explanation to substantiate the statement.

What is micro-injection?

Why is the enzyme cellulase used for isolating genetic material from plant cells but not for animal cells?

Describe briefly Chitinase.

Collect five examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base pair rules.

Do eukaryotic cells have restriction endonucleases? Why?

Distinguish between plasmid DNA and chromosomal DNA.

Explain briefly Restriction enzymes and DNA.

Draw a schematic sketch of pBR $$322$$ plasmid and label Ori and rop genes.

What are palindromic nucleotide sequences?

Why is a thermostable DNA polymerase needed in amplification/genetic engineering?

What is elution in the process of separation of DNA fragments?

Who discovered enzyme DNA polymerase?

Which enzymes are used for releasing macromolecules (DNA) from cell envelope?

What are the two sets of primers needed for amplification of gene?

Draw a schematic sketch of $$pBR 322$$ plasmid and label any two restriction sites.

What is the best method for separation of DNA fragments?

Name the scientists who generated first recombinant DNA molecules.

Expand PCR. Mention its importance in biotechnology.

How are restriction enzymes different from the topoisomerases functionally?

What were the two main discoveries that led to the birth of genetic engineering?

How does restriction endonuclease function.

Why must a cell be made 'competent' in biotechnology experiments? How does calcium ion help in doing so?

How is the amplification of gene sample of interest carried out using polymerase chain reaction (PCR) ?

How is DNA isolated from prokaryotic and eukaryotic cells?

Answer in 3 to 5 sentences:
Draw a neat labelled diagram of a typical agarose gel electrophoresis.

Manipulating with nucleic acid is a trend in biotechnology.
(a) Name any one organism used as vector.
(b) What are DNA polymerase ?

(a) Why must a cell be made 'competent' in biotechnology experiments? How does calcium ion help in doing so?
(b) State the role of biolistic gun in biotechnology experiments.

Expand the following and mention one application of each:
(i) PCR (ii) ELISA

Draw a schematic sketch of pBR 322 plasmid and label the following in it.
(a) Any two restriction sites
(b) Ori and rop genes
(c) An antibiotic resistant gene

Explain briefy.
(a) PCR
(b) Restriction enzymes and DNA
(c) Chitinase

Figure representing the reactions associated with Polymer Chain Reaction (PCR).
Name the steps A, B, C in the progress.

Answer in 40 to 80 words:
Explain in brief the separation and isolation of DNA fragments.

Very short answer type.
Selfing is a kind of inbreeding.

Give reasons for the following:
Restriction enzymes are called chemical scalpels.

A. What is the role of Cry I Ac, Cry IIAb and Cry I Ab?
B. (i) Write the sequence of restriction site of EcoRI and give the sequence of sticky ends after EcoRI digestion
(ii) Name the source of EcoRI restriction enzyme

Given is the diagram of agarose gel kept under UV light.
How are the separated DNA fragments finally isolated?

Study the diagram given and answer the question.
What does E represent in the diagram?

Study the diagram given and answer the question.
Why have DNA fragments in band 'D' moved farther away in comparison to those in band 'C'.

Study the diagram given and answer the question.
Identify the anode end in the diagram.

'The basis of genetic engineering is the discovery of the fact that genes can be cut and joined with the help of enzymes."
a. Name the enzyme used for cutting genes.
b. Name the enzyme used for joining the genes.

Observe the table in which size of different DNA fragments are given and answer the questions:
DNA fragments $$A$$ $$B$$ $$C$$
Size (in Base Pairs) $$700$$ $$1500$$ $$3000$$
Explain the process of separating the DNA fragments.

Observe the table in which size of different DNA fragments are given and answer the questions:
DNA fragments $$A$$ $$B$$ $$C$$
Size (in Base Pairs) $$700$$ $$1500$$ $$3000$$
In the process of separating DNA fragments which fragements moves faster?