Answer: d [Reason:] As the products of RNaseH method and homopolymer tailing method generates dsDNA and blunt ended molecules, there are many methods to attach them to vector. Blunt ended ligation, addition of linkers and restriction enzymes all can be used.

Answer: a [Reason:] The modification of homopolymer tailing method includes modification of primers. Primers can be modified by introduction of restriction sites in them. The 3’ end of first cDNA strand is tailed with C residues. Another oligo-dG primer precedes the introduced restriction site and is contained with the double stranded oligonucleotide region. It is used for second strand synthesis.

Answer: a [Reason:] Double stranded oligonucleotides are required for the synthesis of second cDNA strand. This double stranded oligonucleotide is synthesized by separately synthesizing two strands and then annealing them.

Answer: c [Reason:] For homopolymer tailing of cDNA strands, the blunt ended double stranded molecules are treated with terminal transferase and dCTPs. This leads to addition of C residues at 3’ end. Vector is also treated with terminal transferase and dGTPs. This leads to annealing of vector and cDNA molecules and DNA ligase is not required. The gaps created can be repaired by physiological processes once the recombinant molecules enter the host.

Answer: a [Reason:] As the RNA is non polyadenylated, oilgo-dT primer can’t be used and in place of it a collection of chemically synthesized oligonucleotides is used as primers. They are usually hexamers and are made by equal quantities of A, G, C and T. And thus all hexameric sequences can be synthesized. The primers can attach to the RNA sequences throughout.

Answer: b [Reason:] If the molecules smaller than the fragments required for making a full genomic library are used for collection, this collection is called as shelf. It is a subsection of library.

Answer: b [Reason:] For the construction of a subsection of library, some steps are followed. The size of a particular restriction fragment on which gene is located is known at times. The size of the fragment can be known by carrying out southern blotting. After the size is known, another digest of the genomic DNA is carried out and is done by the same enzyme. DNA fragments of approximately same size are recovered from the gel after carrying out gel electrophoresis. They can be further cloned into a vector.

Answer: a [Reason:] Any cDNA library would represent a fraction of RNA species of an organism, the whole organism can’t be represented in a library. It depends on the developmental stage, physiological state and the tissue from which RNA was isolated.

Answer: b [Reason:] Housekeeping genes are those genes which are present in all the organisms. cDNA libraries may therefore contain housekeeping genes and genes specific to that organism.

Answer: b [Reason:] RNA fractionation is carried out and the basis is size. It is fractioned by size after carrying out separation on oligo-dT cellulose. A sucrose density is used for size based separation. The RNA is applied to the top of a pre-poured gradient and during centrifugation larger molecules move down the tube faster. There are different bands formed on the basis of size in the sucrose density gradient.

Answer: b [Reason:] RNA fractionation is carried out and it is followed by translation of each band in vitro. It is carried out in wheat gram or lysate of rabbit reticuloycte cells. Ribosomes, tRNAs are added in order to carry out the translation with low background. An amino acid is radioactively labelled and thus the polypeptide sequence synthesized is labelled.

Answer: a [Reason:] The analysis of polypeptides after addition of mRNA can be carried out by addition of antibodies. But it is not simply based on antigen-antibody reaction. A substrate for easy precipitation is also added. For this, protein A-Sepharose is added. Protein A-sepharose binds to IgG antibodies. After carrying out centrifugation, it can be pelleted easily.

Answer: a [Reason:] After the recovery of pellets has been carried out by the use of antigen- antibody reaction, denaturation and gel electrophoresis in SDS-Polyacrylamide gel. The amount of radioactivity gives the amount and location of polypeptides.

Answer: c [Reason:] Insertion vectors are the simple vectors and DNA is inserted through a single restriction site. It must be a non-essential gene to maintain phage viability. There is an upper limit on the size of the DNA to be packaged thus only a few kilobases of the extra DNA can be included. Lambda gt10 and gt11 are an example of insertion vectors.

Answer: a [Reason:] Lambda gt10 is having an EcoRI site which contains the cI repressor gene. It accepts an insert of size upto 7.6 kbp which is generally more than that of the size generally accepted by a wild type phage.

Answer: b [Reason:] The presence of insert leads to inactivation of cI gene and this is the method to detect the presence of an insert.

Answer: c [Reason:] If a portion of phage is removed and in its place the DNA of interest is inserted, it is called as substitution or replacement vector.

Answer: d [Reason:] The central portion of lambda genome is approximately 20 kbp and it is not required for lytic growth. The central portion constitutes of repressor gene and thus it has to be removed for the lysogenic growth to take place.

Answer: c [Reason:] The fragment inserted in the place of the central portion of genome is known as stuffer fragment. It is generated by doing partial digestion an enzyme having a 4bp long recognition site.

Answer: b [Reason:] The substitution vector EMBL4 contains two multiple cloning sites. The two multiple cloning sites flank the region which is to be removed. If digestion is done within the multiple cloning sites, it generates central portion and the right and left portions. The right and left portions separated are known as arms.

Answer: a [Reason:] The central portion should be stopped from religating before the stuffer fragment should be ligated. There are chances that central portion gets religated to the arms.

Answer: c [Reason:] A second enzyme is used in order to avoid the relegation of central portion and the arms. In this method, the central portion is cut with a second enzyme after being separated from the arms. These arms are not cut with the second enzymes. Hence the ends created are different now and the arms won’t ligate to the central portion again. It is not necessary to remove the fragments generated because the chances of having a multimolecular reaction which are required for the formation of original phage are very less.

Answer: a [Reason:] Physical separation is also used at times, it constitutes of using gel electrophoresis or by using centrifugation in the sugar density gradient. In sugar density gradient, the separation is done on the basis of size. The arms are separated from the gel or gradient. Gradient is more efficient than gel, thus gradient is more preferred over gel.

Answer: d [Reason:] Packaging in vitro is used to introduce the lambda. It is more efficient than the normal transformation procedure. Thus, the lambda DNA is incubated with lysate of lambda infected cells in a concatemeric configuration. This lysate constitutes of lambda proteins needed for phage assembly and hence the recombinant DNA is also packed. But there is also a large amount of non-recombinant packaging in the background.

Answer: d [Reason:] Packaging in vitro is basically out by two different strategies. In the first strategy, separate strains with chain-termination mutations in different genes (mostly D and E genes) for coat components are used. Temperature sensitive cI repressor is used and due to induction by heat shock, packaging proteins are produced. But packaging is not carried out in any of the strain because none of the strain is having full complement of proteins to carry out packaging.

Answer: c [Reason:] Chain-termination mutations in coat proteins genes are carried out, these genes are D and E genes.

Answer: c [Reason:] Plasmids are double stranded and they are circular in nature. They are capable of replication in bacteria. Insertion of DNA into plasmid allows it to be propagated in host cells and the molecules which are used for propagation by this method are called as vectors.

Answer: d [Reason:] Plasmid generally consists of characteristics such as multiple cloning site, origin of replication, antibiotic resistant genes and beta galactosidase genes. An origin of replication is necessary for the replication to take place.

Answer: d [Reason:] bla is an ampicillin resistant gene. Antibiotic acts by blocking the cross-linking in the bacterial cell wall and thus leading to lysing of the cells. If antibiotic resistant gene is used, which encodes for beta lactamase enzyme it inactivates the beta lactam gene by hydrolysing it.

Answer: a [Reason:] lacZ gene encodes for beta galactosidase enzyme. This enzyme is responsible for cleaving of disaccharides into monosaccharides. It cleaves a substrate known as X-gal, which then liberates a blue dye.

Answer: c [Reason:] When a cloning experiment is carried out, there various orientations which are possible. The orientation which is not propagated to the later stages is linear molecule. Other possible orientations are relegation of plasmid, intramolecular ligation of genomic DNA. Intermolecular ligation of two DNA plasmids and genomic plasmids is also possible.

Answer: b [Reason:] After cloning experiment is carried out, there are various orientations possible. Molecules having combination of new sequences which were not present earlier are called as recombinants.

Answer: a [Reason:] Insertion of DNA into lacZ gene may lead to disruption of the gene function. It always doesn’t so but there are minimal chances that function is not disrupted because often it leads to shifting of the reading frame.

Answer: d [Reason:] Transformation efficiency is defined as the ratio of transformed colonies to that of the amount of plasmid DNA which is taken. It is often altered by altering the competence of the cells.

Answer: a [Reason:] Only cells that have taken up the plasmid DNA would be able to grow. It is so because only plasmid is having antibiotic resistant genes and the rest will die after few generations.

Answer: a [Reason:] The Cre recombinase protein of bacteriophage P1 mediates site-specific recombination at a 34 bp sequence, loxP.

Answer: a [Reason:] For cre-lox excision, the chromosomal copy of the target gene is replaced by target gene flanked by loxP sites. The second step is supply of cre recombinase. Integration of cre is done by a controllable promoter which is followed by the induction of promoter. This induction results in expression of cre and recombination along loxP sites, leading to excision.

Answer: a [Reason:] Generally cre is introduced with the help of a promoter. But it can also be introduced by crossing it with a strain containing the gene or by infection with virus containing it.

Answer: b [Reason:] As the recombination has taken place, the loxP sequence remains there. The remaining loxP sequence has no effect on the surrounding genes.

Answer: c [Reason:] The ability to control the expression of cre allows when and where excision takes place. Specific excision of the target takes place if expression is tissue or time specific.

Answer: c [Reason:] Excision of DNA flanked by loxP sequences is called as floxing at times. Other site specific recombination systems are also used.

Answer: d [Reason:] Any RNA molecule with catalytic activity is termed as ribozyme. Some of the catalytic activity can be removal of introns by self-splicing.

Answer: a [Reason:] Hepatitis delta virus, which is associated with hepatitis B is capable of self cleavage. Ribozymes mainly comprise of RNA molecules which are capable of self cleavage.

Answer: a [Reason:] In the self cleavage reaction, it is an internal nucleophilic attack reaction by 2’ hydroxyl of RNA. The attack is done on the phosphate group in the sugar phosphate group.

Answer: b [Reason:] The self cleavage reaction takes place in RNA. It is because it involves nucleophilic attack by 2’ hydroxyl, which is found in RNA and not in DNA.

Answer: d [Reason:] HGPRT deficient cells in the medium containing all three hypoxanthine, thymidine and aminopterin. It is so because aminopterin blocks endogenous synthesis of purines needed for synthesis of nucleic acid.

Answer: b [Reason:] Presence of wild type in HGPRT—in the presence of calcium phosphate led to DNA uptake and stable transformation.

Answer: a [Reason:] Cells deficient in thymidine kinase are also killed in HAT medium. It is so because pyrimidine synthesis is also blocked in the presence of aminopterin and utilization of thymidine in the HAT medium requires a functional TK.

Answer: b [Reason:] Hygromycin is used as a protein synthesis inhibitor. It is conferred by a bacterial hph gene which encodes hygromycin phosphotransferase. Resistance to hygromycin is used as a selectable marker.

Answer: c [Reason:] Puromycin is a protein synthesis inhibitor and is conferred by streptomyces gene. It does so by encoding puromycin-N-acetyltransferase.

Answer: a [Reason:] Resistance to bleomycin (zeocin) is used as a selectable marker for mammalian cultured cells and it is a DNA damaging agent. It does so by expression of a binding protein.

Answer: d [Reason:] Resistance to methotrexate, which is used as a selectable marker inhibits DHFR enzyme. This enzyme is involved in synthesis of one carbon units and is required for nucleoside synthesis.

Answer: a [Reason:] Histidinol dehydrogenase is also used as a selectable marker. It allows synthesis of histdidine from exogenous histanol and protects from toxic effects of histidinol.

Answer: b [Reason:] Many mammalian cells contain thymidine kinase, the mammalian enzyme uses the analogue less efficiently than the viral enzyme. If cells lack viral enzyme they are resistant to analogue and are able to grow in mammalian enzyme.

Answer: d [Reason:] There is a wide range of host cell lines available. Commonly used human cell lines are HeLa 293T, obtained from kidney and Jurkat which is obtained from lymphoblast from a leukaemia patient.

Answer: b [Reason:] There are various methods for uptake of transformation DNA such as electroporation and DEAE-dextran method. It is a modified polysaccharide and is positively charged. It forms a complex with negatively charged DNA and is taken into cells by endocytosis.

Answer: b [Reason:] Ghosts are those cells whose contents have been removed and replaced by swelling and shrinking in solutions of suitable osmotic strength. Red blood ghosts are also used for introduction of DNA into mammalian cells.

Answer: c [Reason:] In non-permissive cells, virus replication doesn’t take place and viral DNA can be expressed though.

Answer: b [Reason:] SV40 is a virus and it produces two transcripts. These transcripts are produced by early and late transcripts.

Answer: a [Reason:] Splicing of both the transcripts is necessary and it is for efficient expression. Sequences that have not been through splicing process are not expressed efficiently even if introns are removed.