CHAOTIC PCR

NORVALC at QUCDN.QueensU.CA NORVALC at QUCDN.QueensU.CA
Thu Oct 1 10:25:57 EST 1992


   I am frustrated by what I believe is a common experience to PCRers.  I
amplify mtDNA segments from total genomic DNA.  I follow a ds amplification by
ss amplification, clean up and sequencing with Sequenase.  However, apparent
successful amplification and cleanup of the desired fragment, as evidenced
by test gels, is not a good predictor of the eventual success in sequencing
the product.  I find this extremely frustrating.
   As we all know, many variables can affect the outcome of PCR (ie. primers,
contamination, reagent conc. etc.).  Even when I perform PCR under seemingly
identical conditions, I cannot guarantee consistent success.  My questions to
the net are...
1)  What is the most critical determinant of sequencing success in PCR and
what are teh most useful predictors of success?
2)  If there are no useful predictors, is this because the steps involved in
PCR are a higher order process, and am I seeing CHAOS in action?
   I hope that the latter is not the case.  I would like to achieve consistent
results.  I'll provide a summary if this generates a discussion.  Thanks in
advance.

Barry Norval Campbell
Queen's University
Kingston, Ontario, CANADA



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