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I was wondering why establishment of cDNA library required the step of reverse transcription (i.e. turning the sequence into DNA) why not directly using the extracted RNA for sequencing and converting the U to T base? Why bother to have one more step? super super confusing

patpat
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  • because you are stabilising a cDNA library of different expressed products, which you might want to sequence and amplify which is perhaps easier if the product is a DNA? – Behzad Rowshanravan Aug 22 '14 at 14:23

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The construction of a cDNA library involves making a DNA 'copy' of the mRNA because the aim is to synthesise a large set of recombinant plasmid molecules, each containing a different cDNA and each propagated in E. coli.

The heyday of cDNA library construction predates the invention of PCR.

The key difference between the two approaches that you are comparing is that one (cDNA library) involves creation of a physical representation of the expressed genome whereas the other is an informational representation.

Alan Boyd
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  • oh I see I see thank you so much, but then i have come up with another question...mmm..so sorry i have quite a poor background about this aspect....i was wondering why PCR is only applied to DNA why not RNA? – patpat Aug 22 '14 at 14:44
  • Oh n also I was wondering what the physical representation means? – patpat Aug 22 '14 at 14:49
  • I mean that a cDNA library exists as something tangible, not just in a computer. – Alan Boyd Aug 22 '14 at 15:01
  • PCR is carried out with a DNA polymerase, which can only deal with DNA as a template. The exception to this is of course reverse transcriptase, but that can't be used in a PCR because it isn't thermostable and it can't copy the DNA copies that it makes from an RNA template, so exponential amplification cannot take place. – Alan Boyd Aug 22 '14 at 15:04