Are kinetics of oligo:oligo hyb'n different?
deborah.hailstones at agric.nsw.gov.au
Thu Apr 13 23:09:05 EST 2000
I have designed a semi-nested RT-PCR which also includes an additional
anti-sense primer in the second round (based on Sanders and Thomas,
1997). This primer is the rev.compl. of the out-nested primer used in
round 1. S&T97 suggest (and I believe) that it hybridises to the
out-nested primer in the second round, "removing" it from the reaction
and increasing the efficiency with which R2 product is generated (it
certainly does that!).
One of my reviewers, however, has said that "Kinetics of hybridisation
would preclude the removal of one oligo by another in the short cycle
time" (15 sec for annealing).
If this were true, wouldn't the "kinetics of hybridisation preclude"
the binding of the oligo to the template itself (ie, no PCR) ? The
antisense oligo is also present at 2.5 times the concentration of the
sense oligo from R1.
Am i missing something? Is oligo:oligo hybn totally different to
oligo/fragment hybn? If so, how and why?
Any thoughts much appreciated. Cheers
More information about the Methods