The typical procedure to obtain cDNA from genomic extracted DNA is the following:

RNA is extracted from desired tissue, RNA/DNA hybrid is obtained by virtue of reverse transcriptase RNA dependent DNA polymerase, H RNAse creates gaps in RNA of DNA/RNA hybrid and with the aid of a typical DNA polymerase dsDNA is transcribed, right?

Then, the newly synthesized cDNA is linear (like a "small chromosome")? How is it protected from the nucleolytic activity? Is the procedure really like I described or does the reverse transcriptase already give a dsDNA from single stranded mRNA?


1 Answer 1


Here is the rough procedure that I've used to prepare cDNA before

  1. Extract RNA from desired tissue (select for mRNA using the polyA tail of mRNA)
  2. Conduct reverse transcription. This requires a primer complementary to the polyA tail to get the reverse transcriptase started (Edit:You could also use random primers to prime reverse transcription. This eliminates the redundant polyT sequence from the cDNA. This is the method used in the Illumina TruSeq high throughput sequencing library prep kit). Now you have a collection hybrid RNA/DNA double strands
  3. Digest the RNA strand specifically. This can be accomplished with RNAse H as you mention, or by introducing basic conditions, which degrade RNA much more rapidly than DNA due to the 2'OH of RNA. Now you have linear ssDNA complementary to the inital mRNA templates.
  4. From this point your applications may differ. If you are doing microarrays, all you need is ssDNA, since you want to hybridize the cDNA to the array. If your downstream use of the cDNA requires dsDNA, then you need to synthesize a complementary strand to the cDNA (and potentially amplify the product). This can be accomplished through PCR random primers, or some more complicated PCR that will incorporate extra sequences on either side of your cDNA (e.g. bar-codes and adapters in RNA-Seq).

How is [the cDNA] protected from the nucleolytic activity?

If you are digesting the product of reverse transcription with RNAse H, that enzyme digests RNA specifically, so your DNA is unharmed. If you are going with an alkaline conditions treatment, then the RNA will be degraded in those conditions much more quickly than the DNA due to the presence of the 2' OH group in RNA which can act as a nucleophile for a self-cleavage reaction.

  • $\begingroup$ so the cDNA will have a poliT tail? $\endgroup$
    – Katz
    Commented Dec 10, 2013 at 14:23
  • $\begingroup$ It depends. If you just have one strand and you prime with polyA tails, then yes. If you synthesize a second strand, however, then you will have a double strand with one side polyA and one side polyT. I also looked up how the Illumina TruSeq library prep kit does this, and they use random primers to prime the reverse transcriptase, so if you did that then you wouldn't necessarily have a polyT tail on your first strand cDNA. This makes sense if you want to maximize the amount of informative (i.e. non polyA/T) cDNA, say, for RNA-Seq. $\endgroup$
    – A. Kennard
    Commented Dec 11, 2013 at 0:06

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