Random hexamer effects on PCR
smori at nmsu.edu
Sat Jan 7 21:41:39 EST 1995
Steve Hopkins (shopkins at fs1.ho.man.ac.uk) wrote:
: In a previous posting I asked about the influence of reverse transcriptase
: reagents in the PCR step. Thanks to those who replied, though it did not
: really resolve the question about why the reagents can cause amplification.
: Having looked a little more at this it seems the presence of random hexamers
: can amplify PCR (i.e. a diluted RT cDNA product, or cDNA from a PCR reaction,
: with extra random hexamers gives more PCR product than where the hexamers
: are diluted out with the cDNA). Can anyone enlighten me as to:
: 1) Why don't random hexamers have some priming activity in PCR? (too short for
Because normally PCR's are done are relatively high temps. This prevents
the binding of the 6mers to the DNA and the Taq preferentially to the sequence
specific primers present in the reaction mixture.
Also the majority of 6mers have been used for cDNA synthesis step and upon
dilution in your PCR mixture there is even less 6mers available to bind to
: 2) How might random hexamers amplify PCR?
I don't know if the binding of primer is length dependant, but if it is
not I could see 6mers amplifying your DNA is a) they are present at high 's
as to over come your gene specific primers AND b) the reaction is done at
temperatures below 45C.
Shahram Mori _/\_
Program in Molecular Biology _\ /_ Saskatoon/SK/CANADA
Dept. of chemistry and Biochemistry Box 3C \_ _/
NMSU Las Cruces NM ||
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