PCR optimization

vagos el36 at le.ac.uk
Mon Jan 16 22:46:00 EST 2006


hello everyone, I would greatly appreciate some help on this one:

I am trying to optimize a 132mer PCR amplification from template
plasmid DNA. There are two versions of template plasmid that differ in
one specific base pair (shown in capital), as follows:

1 gaattcgagagccctgctcgagctgtAgtggggttcccgagcggccaaagggagcagact
                     >>>>>>>>>>>>>>>

   61 ctaaatctgccgtcatcgacttcgaaggttcgaatccttcccccaccaccacggccgaaa


  121 ttcggtacccggatccttagcgaaagctaagattttttttacgcgtgagctcgactgact
               <<<<<<<<<<<<<<

One version of the plasmid has adenine (A), whereas the other thymine
(T) in its place.

I want to specifically amplify the version that has the adenine (A)
using a complementary Left Primer. Here is my primer design.

OLIGO            start  len      tm     gc%   any    3' seq
LEFT PRIMER         12       19   59.74   63.16    8.00   0.00
ccctgctcgagctgtagtg
RIGHT PRIMER       143      18   59.17  55.56    6.00   2.00
tcgctaaggatccgggta


 Have got any suggestions on how I can achieve complete amplification
of the plasmid with the adenine (A) even if this version is in lower
amount in the plasmid mixture?

I've tried the following methods for achieving specificity:
Increase the annealing temp, decrease the annealing time, primer
booster technique, formamide, and hot start technique.

I've managed to achieve ~ 60% specificity according to agarose gel
electrophoresis. Have you got any ideas on how I could improve on this?
Please note that I cannot use nested primers, because of the nature of
my work.

Any ideas welcome.
Thank u in advance,
Evagelos Liapis, PhD student



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