Is it Taq or the primer responsible for the mistake?
c557652 at showme.missouri.edu
Sat Mar 4 19:13:17 EST 1995
In article <3j87e8$dm7 at masala.cc.uh.edu> benedik at uh.edu (benedik at uh.edu) writes:
>In article <01HNP6F12GKI00PF24 at Post-Office.UH.EDU
>CPedemonte at UH.EDU (carlosh. pedemonte) writes:
>> 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.
>> Best, carlos
>I agree with your reasoning. Did you receive the computer printout from
>the company for your oligo? I have found on at least 2 occasions (out
>of a few hundred oligos) that the company typed in my sequence
>incorrectly. These were both changes and I found out by carefully
>comparing the printout they sent me with the sequence I sent them.
>So the simplest explanation would be a type.
>Alternately there are possible reasons for a machine failure, such as
>they changed reagents during your run, there was an air bubble in the
>line, etc etc. So it is possible and not all oligos are perfect.
>Michael Benedik benedik at uh.edu
Not so in our case! We recently did a series of constructs using PCR
methods and, unfortunately, Taq. Numerous clones had mutations and an
overwhelming percentage of these were in the primer region! The sequencing,
BTW was done manually with Sequenase. So, we checked with the folks across
the hall who made the primer and their machine print-out was correct. Our
explanation is that Taq is incorporating mismatches to the primer as it
finishes off a piece of DNA. Consequently when the piece is cloned, one
strand is correct (primer) and one is wrong (Taq added DNA). If the wrong
one gets amplified by the bacteria you pick you can have a mutation in the
primer region. Our products were only 200-400 bases long. By sequencing
more clones we eventually found one for each construct that has the correct
sequence. Our mutations rate is severe, but we don't plan to pursue
optimizing our Taq reactions. Instead we are switching to a proofreading
enzyme in the future.
So, although you may have a bad primer, you can also have mutations by Taq
in the primer region.
| Robert Woodward c557652 at showme.missouri.edu |
| Department of Physiology, University of Missouri |
| voice: 314-882-5374, FAX: 314-884-4276 |
| http://www.missouri.edu/~c557652 |
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