Sequencing PCR Products - How Best?

Dr. Peter T. Boag BOAGP at QUCDN.QueensU.CA
Mon Oct 5 09:45:31 EST 1992


This is a repost of a query made a week or so ago. I got some
useful replies, but the original query did not seem to hang
around long. Thanks if you have already replied, and once again
I will summarize when the responses taper off.

                                We sequence mtDNA segments amplified from
total genomic DNA, using a DS amplification followed by asymetric
amplification and SS sequencing with Sequenase. We never have a problem with
DS amplification if the primers are good, but asymetric amplification
and subsequent sequencing have always been unreliable, with a high
failure rate. Quality and quantity of SS template seems the problem
but we have tried every purification technique in the book without
consistent results (precipitation, Millipore and AMicon spin dialysis,
gel slice excision, sepharose spin column, etc.).
 We have been considering alternatives such as
       cycle sequencing, or cloning
of DS PCR product, or lambda exonuclease production of single stranded
template, etc. Any experience from the net would be much appreciated.
We are leaning towards giving cycle sequencing a serious try and would
appreciate experience with various kits available that have been tried
with PCR products. Thanks in advance, I will summarize for NET if
response dictates. - Peter Boag, Biology, Queen's University, Kingston,
Ontario, Canada.



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