This set of Genetic Engineering Multiple Choice Questions & Answers (MCQs) focuses on “Cloning the cDNA and Specialized Libraries – 1”.
1. RNaseH method and homopolymer tailing method generates blunt ended cDNA molecules. Which of the following can be used for attaching them to vector?
a) Blunt ended ligation
b) Addition of linkers
c) Using appropriate restriction enzymes
d) All the methods can be used equivalently
Explanation: 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, the addition of linkers and restriction enzymes all can be used.
2. Choose the correct statement for modification of homopolymer tailing method.
a) It includes modification of primers
b) Primers are varied by simply altering their size by randomly adding or removing bases
c) The 5’ end of the first cDNA strand is tailed with C residues
d) A single stranded oilgonucleotide is then used for second strand synthesis
Explanation: The modification of homopolymer tailing method includes modification of primers. Primers can be modified by the introduction of restriction sites in them. The 3’ end of the 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.
3. By synthesizing two strands separately then annealing them leads to formation of double stranded oligonucleotide.
Explanation: 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.
4. Choose the incorrect statement for the homopolymer tailing of cDNA strands.
a) The blunt ended double stranded cDNA molecules are treated with terminal transferase and dCTPs
b) Vector is also treated with terminal transferase and dGTPs
c) The vector and cDNA can now anneal with the help of DNA ligase
d) If gaps are created they can be repaired by physiological processes
Explanation: For homopolymer tailing of cDNA strands, the blunt ended double stranded molecules are treated with terminal transferase and dCTPs. This leads to the 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.
5. Choose the correct statement if the RNA is non polydenylated.
a) A collection of chemically synthesized oligonucleotides is used as primers
b) They are usually tetramer
c) Unequal quantities of A, G, T and C are used
d) The primers attach at only specific sequences for first strand synthesis
Explanation: 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.
6. In case if molecules smaller than the fragments required for making a full genomic library are used for making a collection. This collection is called as ___________
c) small library
d) mini library
Explanation: If the molecules smaller than the fragments required for making a full genomic library are used for collection, this collection is called a shelf. It is a subsection of the library.
7. Choose the correct statement for construction of a library subsection.
a) The size of a particular restriction fragment on which the gene is located is not known
b) The size of the restriction fragment can be known by carrying out southern blotting
c) Another digest of the genomic DNA is carried out by a different enzyme
d) DNA fragments of different size are recovered after carrying out gel electrophoresis
Explanation: For the construction of a subsection of the 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 the approximately same size are recovered from the gel after carrying out gel electrophoresis. They can be further cloned into a vector.
8. Any cDNA library would represent a fraction of RNA species of an organism.
Explanation: 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.
9. What do we mean by housekeeping genes?
a) Housekeeping genes are those genes which are specific to an organism
b) Housekeeping genes are those genes which are present in all the organisms
c) Housekeeping genes are those genes which are meant for repair and maintenance in a species of organism
d) Housekeeping genes are those genes which required for the replication process
Explanation: 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.
10. Choose the correct statement for RNA fractionation.
a) The RNA is fractioned by size but before separating on oligo-dT cellulose
b) A sucrose density gradient is used
c) The RNA is applied to the top of a pre-poured gradient and during centrifugation smaller molecules move down the tube faster
d) Different bands are formed according to the density in the sucrose density gradient
Explanation: RNA fractionation is carried out and the basis is the 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.
11. What is done after RNA fractionation is carried out?
a) Each band is translated in vivo
b) Translation is carried out in wheat gram or lysate of rabbit reticulocyte cells
c) Translation is carried out with a high background
d) Amino acid is not radioactively labelled
Explanation: RNA fractionation is carried out and it is followed by a 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.
12. The polypeptides produced after addition of mRNA are analysed with antibodies. Choose the incorrect statement for this analysis.
a) Antibodies are added to each reaction tube and precipitation is simply based on antigen-antibody reaction
b) Along with simple antigen-antibody complex, a substrate is added for easy precipitation
c) Protein A-Sepharose is added
d) Protein A-Sepharose binds to IgG antibodies
Explanation: The analysis of polypeptides after addition of mRNA can be carried out by the 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.
13. What is done after the recovery of pellets has been carried out in order to know the amount of polypeptides?
a) Denaturing and gel electrophoresis in SDS- Polyacryamide gel
b) Gel electrophoresis in agarose gel
c) Quantitative PCR
d) Weighing pellets
Explanation: 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.
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