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.

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.

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.

Explanation: The loop region in the hairpin structure is single stranded in nature. The single stranded portion can be cleaved by S1 nuclease.

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.

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.

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.

Explanation: The first primer in the case of 3’ RACE is oligo-dT adaptor molecule. The second primer is an internal sequence.

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.

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.