Rules of thumb for sequencing cloned PCR products?

Mayumi Yagi myagi at u.washington.edu
Tue Aug 27 11:41:18 EST 1996


Hi,

Our lab has done a lot of sequencing of PCR products, in some cases
looking for mutations in a particular disease state.  In spite of the
stated error rate (which I remember as being more like 10e-4), we haven't
really seen many Taq-induced errors; the error rate from the automated
sequencer is higher than that from the PCR.  In the case of the postdoc
looking for the mutations in patient DNA, he routinely sequenced eight
individual clones from each PCR cloning.  I belive that, in sequencing
a total of about 5 kb each from ten people (from a couple of different
families), he saw only two PCR errors.

As for the sequence you have in hand, our experience would indicate that
the sequence you have is more than likely error-free.  I think sequencing
one or two other clones would be more than sufficient to satisfy most
reviewers.

Hope this helps.
------------------------------------------------------------------------------
Mayumi Yagi, Ph.D.
Research (151)
Veterans Affairs Medical Center
1660 South Columbian Way
Seattle, WA 98108
Tel (206)762-1010, ext 1918
Fax (206)764-2598






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