Rules of thumb for sequencing cloned PCR products?

Mark Pallen m.pallen at ic.ac.uk
Tue Aug 27 10:37:08 EST 1996


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. 

I remember reading somewhere that a rule of thumb was that you are 
likley to get one misincorporation error for every 1.5 kb that you 
amplify. If true, my colleague would have a rougly fifty:fifty chance 
of having the right sequence. I also remember from somewhere that 
doing three separate PCRs and cloning then sequencing all three was a 
reasonable way to get round the Taq/PCR-induced error problem. My 
colleague is doing that at the moment.

What I wanted to check is whether there is a consensus on what is 
acceptable here. Leaving aside the question of whether one shouldn't 
use a proof-reading enzyme instead of Taq (she tried Vent and couldn't 
get it to work), what do people think of my vaguely remembered rules 
of thumb? If she were to submit the original sequence (the one derived 
from one clone from one reaction) for publication and/or in her thesis 
and *you* were the referee or examiner, what would you do about it? 
What do you think are reasonable rules of thumb here, and what 
literature exists to back them up?

Cheers

-- 
Mark
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Dr Mark Pallen, Senior Lecturer in Medical Microbiology,
St Bartholomew's Hospital Medical College, London, EC1A 7BE
currently on a Research Leave Fellowship at Imperial College 
Rm 502, Dept of Biochem, Imperial College, London, SW7 2AY
email:m.pallen at ic.ac.uk  WWW: 
http://www.qmw.ac.uk/~rhbm001/mpallen.html
phone: day ++44(0)1715945254, eves ++44(0)1815057937, FAX 
++44(0)1715945255
Author, Microbial Underground: http://www.qmw.ac.uk/~rhbm001
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"Nothing in biology makes any sense except in the light of evolution"
T. Dobzhansky
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