Rules of thumb for sequencing cloned PCR products
dspinella at chugaibio.com
Tue Aug 27 10:44:28 EST 1996
Your question inspired me to do a litle research. My lab has been
cloning PCR products (RT-PCR) derived from ligand binding domains of
single-chain receptors (cytokine receptors, growth factor receptors,
etc.). The PCR products are typically 600 -1600 bp in length. Our
standard is to pick 5 clones from each PCR product and sequence them
all. To date we have cloned nearly three dozen PCR products in this
way -- more than 150 templates sequenced. Because the sequences are
already published in the literature, we can compare our sequences to
the published ones to look for Taq misincorporations.
What we have found is that, on average, we need to sequence 3-4 clones
to find one "clean" one. The "mutations" are typically different in
each incorrect clone which is our clue that we are truly dealing with
a Taq misincorporation. Occasionally, we find that all five of our
clones has the same base substitution relative to the published
sequence. In this case, we ascribe the "mutation" to an allelic
variant, so we don't count it as a PCR error. Another thing we have
noticed is that PCR "error" rate varies from template to template.
Some of our targets are completely clean in every clone, while others
have a different mutation (sometimes multiple mutations) in each of
the five clones we sequence.
The bottom line is that I suspect that your rule of thumb estimate of
the Taq error rate is probably a little low -- and I certainly
wouldn't trust a single clone to provide an accurate sequence. One
thing you can do (assuming that you are cloning only to obtain
sequence information) is to avoid cloning altogether, and sequence
directly from your PCR product. In this way you get a "consensus"
sequence which is much more likely to be accurate. The ABI protocols
are very efficient at providing robust sequencing data from uncloned
Hope this helps. Regards.
D.G. Spinella, Ph.D.
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