PCR Mistake

Koen De Smet k.desmet at nospam.ic.ac.uk
Thu Apr 2 10:12:03 EST 1998

ddsyr at PUBLIC.EAST.CN.NET wrote:
> Dear Netters:
> I have a general question. When PCR a cDNA of known sequence, from same plant species, but different
> cultivars, the sequence of this cDNA may have some different with the publishing sequence. Then can we
> have any way to jugde that some base different are from the Taq enzyme mistakes, and some are from the
> cultivar different?
> I am appreciate your suggestion.
> Please reply to my email of ddsyr at public.east.cn.net
> Ma

Sequence the PCR product directly, don't clone it. The vast majority of 
the individual PCR molecules will have the right nucleotide at a 
specific position. 
Even if Taq makes a mistake in the first cycle (starting from dsDNA), 
this msitake will only be found in 25% of all the PCR product, because 
you still have the correct forward and reverse strand, one correct 
amplified strand and one that contains the mistake that will be 
amplified in subsequent amplifications. 

Koen De Smet
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     Imperial College School of Medicine at St Mary's   

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