Is it Taq or the primer responsible for the mistake?
carlos h. pedemonte
CPedemonte at UH.EDU
Fri Mar 3 13:22:41 EST 1995
We have sequenced part of a plasmid and found a missing base. Since we
have used PCR with Taq polymerase to produce this part of the plasmid, we
want to identify what have caused this deletion. We think that the problem
is in the custom-made primer. But we would like to hear your opinion, to
be sure that our conclusion is correct.
We used a custom-made primer to produce a fragment by PCR (with Taq
polymerase). The fragment and the vector were cut with restriction enzymes
and ligated. DH5-alpha competent cells were used for transfection.
Colonies were screened by PCR. Two colonies were positive for the presence
of the fragment. These colonies were picked from the plate to do sequencing
(Sequenase PCR with Taq polymerase). In both sequences one base was
missing; this base corresponded to the custom-made primer (not to a sector
that was amplified by PCR). The sequence of one of the colonies was
repeated. Again, the same base was missing.
OUR REASONING: The mistake can be produced by either i) Taq polymerase, or
ii) the primer has a missing base. Taq polymerase could only have produced
a mistake during the sequencing. THE MISTAKE CANNOT HAVE BEEN PRODUCED
DURING THE PREPARATION OF THE FRAGMENT BECAUSE THE MISSING BASE IS IN A
SECTOR THAT CORRESPOND TO THE PRIMER. Since it is very unlikely that Taq
would produce three times the same mistake, we concluded that the primer is
wrong. The mistake was done during the preparation of the primer.
1) IS OUR CONCLUSION CORRECT?
2) Can oligonucleotide synthesizers produce primers that are wrong?
Thank you for your help.
Carlos H. Pedemonte Telephone: 713-743-1211 (office)
Dept. of Pharmacology 713-743-1228 (lab)
University of Houston Fax: 713-743-1229
Houston, TX 77204-5515 e-mail: pedemonte at jetson.uh.edu
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