A single stranded DNA or RNA joined with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells. It is followed by detection using autoradiography. The clone having the mutated gene will not appear on the photographic film, because the probe will not have the complementarity with the mutated gene. 

When bacteria (a prokaryote) is engineered to express a gene from a plant (an eukaryote), a slightly  different approach is used. Eukaryotic genes have both exons and Introns which are transcribed into the primary transcript. Primary transcript is then processed by splicing (removal of introns and joining of exons), capping and tailling to produce functional mRNAs. This functional mRNA is then expressed into proteins. Prokaryotes lack such machinery, hence expression of an eukaryotic DNA becomes difficult in prokaryotic cells. Therefore, scientists prepare cDNA from the functional nRNA (without introns) using reverse transcriptase. This DNA is incorporated into bacteria (or another prokaryotic cell) for expression of plant gene. Such foreign gene synthesises a protein with the same sequence of amino acids as in the plant, and therefore the proteins have the same structure and function as in the plant.

So, the correct answer is 'Isolate mRNA from the organism, reverse transcribe and generate cDNA, Insert into a plasmid vector and transform into eukaryotic cells such as yeast. Screen colonies for the ability to glow in the dark'.

Since DNA polymerase requires a free 3' OH group for initiation of synthesis, it can synthesize in only one direction by extending the 3' end of the preexisting nucleotide chain. Hence, DNA polymerasemoves along the template strand in a 3'–5' direction, and the daughter strand is formed in a 5'–3' direction.→→ 3 direction'

Plasmid - generally up to 15 Kb long DNA fragments 
Phage - up to 23 Kb long DNA fragments
Cosmid - up to 45 Kb long DNA fragments
BAC - up to 300-350 Kb long DNA fragments
YAC - up to 1 Mb long DNA fragments

Very low count of bacteria or viruses (when the symptoms of the disease are not yet visible) can be detected by multiplication of their nucleic acid by PCR, (PCR can detect very low amounts of DNA). PCR is usually used to detect HIV in suspected AIDS patients.

A pBR 322 is a plasmid derived from E. coli. It is widely used E. coli cloning vectors. It contains the origin of replication and the rop gene, which encodes a restrictor of plasmid copy number. It also contains unique restriction sites for more than 40 restriction enzymes. 

Restriction endonucleases cut DNA segments at specific sites of 4 or 6 base pair length, called the restriction sites and are therefore known as the molecular scissors. DNA polymerase helps in the synthesis of DNA and RNA polymerase catalyses the synthesis of RNA. DNA ligases on the other hand help in joining DNA fragments by helping in the formation of phosphodiester bonds between them. So, the correct option is 'restriction endonucleases'.

Bacterial species are the major source of commercial restriction enzymes. These enzymes serve to defend the bacterial cells from invasion by foreign DNA, such as nucleic acid sequences used by viruses to replicate themselves inside a host cell. Basically, the enzyme will chop DNA into much smaller pieces which pose little danger to the cell. The enzymes are named for the species and strain of bacteria that produces it. For instance, the first restriction enzyme extracted from Escherichia coli strain RY13 is called EcoRI, and the fifth enzyme extracted from the same species is called EcoRV.

Restriction enzymes, also called restriction endonucleases, bind to DNA and cleave the double strand, forming smaller pieces of DNA. There are three types of restriction enzymes; Type I restriction enzymes recognize a DNA sequence and cut the strand randomly more than one thousand base pairs away from the site. Type II restriction enzymes, the most useful for molecular biology laboratories, recognize and cut the DNA strand predictably at a specific sequence which is usually less than ten base pairs long. Type III restriction enzymes are similar to Type I, but these cut the DNA about thirty base pairs from the recognition sequence.

The plasmid is a naturally occurring, extrachromosomal, circular, double-stranded DNA that can replicate autonomously. It has been manipulated to act as vector for the biotechnological processes. 

Polymerase chain reaction (PCR) is a technique that exponentially amplifies a single copy of a specific DNA segment. It generates thousands of copies of that specific DNA segment. After completion of one cycle, 2 copies are produced from a single DNA segment. After completion of the second cycle, 2$$^2$$ = 4 copies are produced. Similarly, after n$$^{th}$$ cycle, 2$$^2$$ copies are produced, where n is the number of cycles. Hence, after completion of 6 cycle, 2$$^6$$ = 64 copies will be produced. 

A restriction enzyme or restriction endonuclease 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.

Correct Answer: A

Correct Answer: B

• For delivering the foreign DNA into the recipient cells we take the help of genetic vectors. They can replicate autonomously and typically include features to facilitate the manipulation of DNA. They are also referred to as the vehicle for foreign DNA.

a) The first restriction endonuclease enzyme, which has been isolated and characterized was Hind II. It was isolated from the bacterium Haemophilus influenzae by Hamilton Smith, Thomas Kelly and Kent Wilcox. 

A DNA sequencing reaction was performed with the fragment 5−XXXGCGATCGYYYY−3′ as the template, dideoxy GTP, all the four dNTPs, and the required primers and enzyme. XXXX and YYYY in the given DNA fragment represent primer binding sites. The set of fragments obtained during the reaction will be CGATCGC, because if primer is attache to the XXXX binding site it polymerization starts and its complimentary fragment is CGATCGC.

Most important part if Ti-plasmid at which desired gene is put to target into the plant cell is Vir gene. The Ti-plasmid contains several genes containing Ti-gene, Vir gene etc. Ti gene is used to express specific compounds, which are used by the bacterium as a carbon source. The Vir gene is an important part of  Ti-plasmid at which desired gene is put to target into a plant cell.

A bacterial plasmid is typically circular and double stranded. It is also called extra chromosomal DNA that can replicate in a cell separately from the chromosomes.

Plasmids are small, circular, extrachromosomal DNA which are able to self replicate. They are seen in certain bacteria. They usually contain some unique phenotypic characters to some bacteria like resistance to antibiotics. They are widely used in genetic engineering for transfer of desired gene. The transformants are identified due to presence of antibiotic resistance genes.

Heat Shock is given to bacteria for transformation that alters the membrane fluidity. The increase in temperature creates pores in the bacterial plasma membrane and allow plasmid DNA to enter the bacterial DNA. For the heat shock, the DNA-bacteria mixture is put into a 42C to 37C water bath. 

Vectors are the agent that helps in transporting the DNA to the host cell for multiplication of the alien DNA.

Restriction endonucleases are a class of enzymes that cut the DNA by recognising a specific sequence or base pairing of the DNA.

pBR322 is an important cloning factor which adds the DNA to the host cell. The pbr322 vector is a cloning vector for E.coli and it contains restriction sites for many restriction endonucleases such as Bam HI and EcoRI. This also has various cloning sites as well as selectable markers. These vectors are generally single-stranded DNA molecules and they have the origin of replication sites which enables the replication of any piece of DNA when this is linked to the host cell.

The correct answer is 'A-Eco RI, B- Hind III, C- Bam HI, D-Sal I'.
Option B is correct.

• Agarose is derived from seaweeds Gelidium and Gracilaria
• Opine synthesis is the function of Ti plasmid of Agrobacterium
• Biolistic is the term used for gene gun used for biotransformation
• Thermocycler is used to carry out PCR reaction

So the correct option is B. (I−D,II−C,III−B,IV−A)

Vectors, gene gun ( biolistic ) and microinjection are all viable techniques to insert foreign DNA into the host cell. So, the correct option is All of these.

Insertional inactivation is the inactivation of a gene upon insertion of another gene inside in its place or within its coding sequence. This helps the selection of transformant and as well as recombinant colonies in selective antibiotic medium aided due to insertional inactivation of the antibiotic gene which becomes inactive due to foreign gene insert within its sequence. 

The areas of application of PCR include 

The development of molecular techniques that access viral load and the development of genotypic resistance have revolutionized the treatment of HIV disease. Commercially available viral load assays use a number of different approaches from reverse transcriptase PCR to amplification of branched chain DNA. New real-time PCR assays are under development, including LightCycler- and TaqMan-based tests. So, the correct answer is option A. ( X=PCR ).

• Agrobacterium tumefaciens is a pathogen of several dicot plants. It delivers a piece of DNA known as T-DNA' in the Ti plasmid which transforms normal plant cells into tumor cells to produce chemicals against pathogens. 
• The restriction enzyme Eco Rl, is isolated from Escherichia coli RY13. The first restriction enzyme Hind II was isolated from the bacterium Haemophilus influenzas. 
• The restriction enzyme Bam HI is isolated from Bacillus amyloli. 

So, the correct option is option C.

There is an intimate connection between genes and synthesis of polypeptides or enzymes The relationship between the sequence, of amino acids in a polypeptide and nucleotide sequence of DNA or mRNA is called genetic code. To code a polypeptide of 162 amino acids 486 nucleotides required but here, methionine is absent. Therefore, 489 nucleotides required and methionine may remove from polypeptide later.
So, the correct option is option C.

• In microinjection, a glass-made micropipette is used for DNA transfer.
• The chemical-mediated genetic transformation uses chemical agents.
• In Electroporation, the target cells are mixed and treated with an electric field to create pores for DNA entry.
• In the biolistic method or gene gun method, the foreign DNA is precipitated over the surface of metals like; gold or tungsten, and bullets are prepared.
• These bullets are then fired into the solution of target cells. This foreign DNA gets inserted and helps in the production of desired products.

So, the correct option is option D.