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Specificity of PCR primers.

Michelle Gleeson michelle at MOLECULE.BIO.UTS.EDU.AU
Wed Oct 9 17:25:27 EST 1996

Hi Zhongtang,

Where is the single mismatch located?  If it is at the extreme 3` end of
the primer you MIGHT be lucky to get priming to only your desired
target, if the conditions you use are very stringent.  Otherwise, there is no
 hope - PCR is quite tolerant of mismatches between target and primer at any
other position.  I have had primers bind where only the first 4 3' and
last 4 5' bases matched the target, with nothing but mismatches in the
middle :-(

Good luck,

Michelle Gleeson
Molecular Parasitology Unit
University of Technology
michelle.gleeson at uts.edu.au

On 8 Oct 1996, Zhongtang Yu wrote:

> I am trying to design a PCR primer for a specific 16S rRNA.  I wonder if a
> primer (a 20mer) which has complete match with my target but only ONE
> mis-match with other related 16S rRNAs can be use to amplify only my target.
> My rRNA will be from mixed microbial community.  Any info will be
> appreciated.
> Zhongtang Yu
> Dept. of Microbiol. UBC.

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