Rules of thumb for sequencing cloned PCR products?
bernard at elsie.nci.nih.gov
Thu Aug 29 16:55:13 EST 1996
In article <32231624.7189 at ic.ac.uk>, m.pallen at ic.ac.uk says...
>Perhaps you fellow netters can help with a problem in our lab
>concerning what's acceptable and what's not in the sequencing of
>cloned PCR products?
>A PhD student in the lab has sequenced a c. 800 bp DNA fragment that
>was amplified from E. coli then cloned into a plasmid. The sequencing
>was done on an ABI automated sequencer. Using five different primers
>she got the whole sequence covered well enough to assemble a contig
>(i.e every base sequenced on both strands and in cases of ambiguity,
>sequenced at least three times).
>After she had finished, I pointed out that, as she had used Taq in the
>initial PCR, even though we were happy that the sequence we had was
>indeed that of the cloned PCR product, this sequence might not be
>identical to that on the E. coli chromosome as only *one* clone
>derived from *one* PCR had been sequenced, and there was a chance that
>Taq-induced misincorpations had occured in the PCR.
Is the sequence expressed in e.coli to a reasonable level? If so
you could test for the presence of mutation(s) by RNase protection.
Do a labelled run off transcription of the cloned sequence, anneal
to e.coli RNA and digest. I guess this is somewhat similar to the
method used in Ambion's mismatch detection kit. If the sequence
is not expressed you could try a similar method with S1 nuclease
and genomic DNA.
In any event I'm glad you question the Taq fidelity.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
More information about the Methods