Are kinetics of oligo:oligo hyb'n different?

Deborah Hailstones deborah.hailstones at agric.nsw.gov.au
Thu Apr 13 23:09:05 EST 2000


     Hi all,
     
     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
     
     Deb

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