This set of Genetic Engineering Multiple Choice Questions & Answers focuses on “cDNA Libraries, Polyadenylated RNA and cDNA Synthesis – 2”.
1. Choose the incorrect statement for second DNA strand synthesis.
a) The RNA: DNA complex is treated with the enzyme RNaseH and DNA pol
b) The enzyme RNaseH is responsible for nicking the RNA strand and leaving free 3’ hydroxyl ends
c) These RNA fragments can be used as primers
d) There is no portion of RNA left attached to the DNA strand
Explanation: For second strand DNA synthesis, the RNA: DNA complex is treated with RNaseH enzyme along with DNA pol. The enzyme is responsible for nicking the RNA strand and leaving 3’ hydroxyl ends. These free ends are used for second strand synthesis. These RNA fragments can be used as primers. After DNA: DNA duplex formation, only a small portion of RNA is left at the 5’ end.
2. What is the final product of the RNaseH method?
a) blunt ended dsDNA
b) staggered dsDNA at both ends
c) staggered dsDNA at 3’ end
d) staggered dsDNA at 5’ end
Explanation: The final product of the RNaseH method is blunt ended dsDNA. The RNA piece left at the 5’ end is removed by RNase and thus blunt ended dsDNA is left.
3. What would not happen if the RNA strand is completely removed from RNA: DNA hybrid?
a) There are no chances of the synthesis of the second DNA strand
b) Chance complementarity would take place
c) Hairpin structure would be formed
d) Hairpin structure is formed is not the final structure
Explanation: If the RNA strand is completely removed from RNA: DNA hybrid, the single stranded DNA remains. Then chance complementarity takes place and the 3’ end complements with any other portion in the single stranded DNA. It leads to the formation of a hairpin structure and the 3’ end extends in the presence of DNA pol and nucleotides.
4. The loop region is single stranded. It can be cleaved by using which enzyme?
b) S1 nuclease
Explanation: The loop region in the hairpin structure is single stranded in nature. The single stranded portion can be cleaved by S1 nuclease.
5. Choose the correct statement with respect to the self priming method of cDNA synthesis.
a) It is less preferred than RNaseH method
b) A hairpin structure is formed with guarantee
c) The sequence corresponding to the 5’ end is lost
d) Reverse transcriptase is not used
Explanation: The self priming method is based on the formation of a hairpin structure. But there are only chances and no guarantee of formation of hairpin structure. In the first strand synthesis, reverse transcriptase is used. The sequence corresponding to the 5’ end is lost and in cDNA large deletions are observed. This method is less preferred than RNaseH method.
6. Choose the incorrect statement for the method homopolymer tailing.
a) The first step is the RNA: DNA hybrid synthesis
b) Terminal transferase is used for the addition of nucleotides on 3’ end
c) Terminal transferase adds only at DNA strands
d) The DNA strand is now having known sequence at 3’ end
Explanation: Homolpoymer tailing is also used for cDNA synthesis. The first step remains same as the other methods, which is the synthesis of RNA: DNA hybrid. It is then treated by terminal transferase, the enzyme is responsible for adding nucleotides on 3’ end of both DNA and RNA. The 3’ end is now having a known DNA sequence and it is used as a tail in the reaction.
7. Choose the correct statement for RACE.
a) It stands for Random Amplification of cDNA ends
b) It is for cloning particular cDNA ends
c) It is only of one type, which is 5’ RACE
d) Sequence data is not available in any case
Explanation: RACE stands for Rapid Amplification of cDNA ends. Sometimes, we wish to clone specific cDNA portion and are having some sequence data. It is meant for cloning specific cDNA ends and is classified into two types, 5’ RACE and 3’RACE.
8. The first primer in the case of 3’ RACE is ___________
a) internal sequence
b) oligo-dT adaptor molecule
c) oligo-dA adaptor molecule
d) adaptor oligo-dT primer
Explanation: The first primer in the case of 3’ RACE is oligo-dT adaptor molecule. The second primer is an internal sequence.
9. The first cDNA strand in 5’ RACE is tailed with oligo-dA tail.
Explanation: The first cDNA strand in the case of 5’ RACE is tailed with an oligo-dA tail. The synthesis of the first cDNA strand is by an internal primer.
10. What is the second primer in the case of 5’ RACE?
a) Internal primer
b) Oligo-dA sequence
c) Adaptor-oligo-dT primer
d) Oligo-dT adaptor molecule
Explanation: The second primer ie the primer used for the synthesis of the second cDNA strand is adaptor-oligo-dT primer. Subsequent PCR is carried out by using internal primer for coding sequence.
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