random hexamers as primers for RT in RT-PCR

Shahram Mori smori at nmsu.edu
Sat Mar 11 22:08:36 EST 1995


Robert Hewitt (rhewitt at HELIX.NIH.GOV) wrote:
: Someone in my lab has found that random hexamers sometimes work better
: than oligo-dT, as primers for RT in RT-PCR.  This seems to be especially
: true when the sequence to be amplified is a long way from the 3' end of
: the message.  Our explantion is that reverse transcription from an
: oligo-dT primer may often not extend as far as a 5' sequence to be
: amplified.  From a random hexamer however, it may stand a better chance.  
: Please let me know if you disagree with this explanation. 

: Can anyone explain what happens when a number of random hexamers anneal to
: the same mRNA, so that reverse transcription occurs from a number of
: primers along the same message?  In this situation, would the multiple 1st
: strand cDNA molecules join up to produce a single molecule?  If this does
: happen, I would be grateful if anyone can provide the explanation. 

: Many thanks,

: Robert Hewitt
You will have a number of fragments of the same gene produced.About them 
joining Unless you have DNA Pol I or some kind of ligase, I don't think 
so. There will be a different number of DNA fragments of different sizes.
I guess it's a race between RT and the primers.
Cheers
 --
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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