Rules of thumb for sequencing cloned PCR products?

Bernard Murray bernard at
Thu Aug 29 16:55:13 EST 1996

In article <32231624.7189 at>, m.pallen at 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  (National Cancer Institute, NIH, Bethesda MD, USA)

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