Efficient PCR with 3' mismatch?
David R. Johnson
johnson at biomed.med.yale.edu
Mon Jun 23 12:54:03 EST 1997
Dear netters,
After RT-PCR amplification of a highly polymorphic gene family (HLA),
we have observed reasonable yields of a product that is mismatched from
the primer at the 3' end nucleotide (even one with the last 3
nucleotides mismatching). The remainder of the primer matches
perfectly. Is this a common occurance? Can anyone suggest ways to
reduce or avoid this problem?
PCR conditions are:
35 cycles of
95oC 30s denature
62oC 30s anneal (20 nt primers, Tm ~62oC)
72oC 30s extend (~1kb product)
template = oligo dT primed, Superscript reverse transcribed cDNA
thanks for any suggestions,
Dave
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