increasing amplification by 2 rounds of PCR

RYBICKI, ED ED at micro.uct.ac.za
Mon Nov 29 02:38:52 EST 1993


> Is nested PCR better than this 2 rounds of PCR with the same 
primers? In this
> case could somebody explains me why?

Speaking as one firmly biased in favour of nested PCR (did it in 
collaboration with my wife, worked well), I must recommend it 
strongly for the following reason:

If you have amplified up crud in your first 35 cycles, you will 
re-amplify it even better in the next 35 cycles using the same 
primers, even if you gel-purify or "stab out" the band of choice, 
and especially if the crud is smaller than the desired band 
(competes out the larger product).

If, however, you use another set of (nested) primers for the next 
amplification, you re-specify the initial specificity due to 
annealing of primer and limiting amounts of target, and any crud 
amplified up in the first round has only a small chance of being 
re-amplified in the second round.  Works very well in terms of 
sensitivity, and also lessens background dramatically: with 
papillomavirus PCR it allowed unequivocal identification of 
positives by EthBr staining of agarose gels, rather than only after 
Southern hybridisations.

See Williamson and Rybicki, 1991, J Med Virol 33: 165-171.
  ____________________________________________________________________
 | Ed Rybicki, PhD             |                                      |
 | (ed at micro.uct.ac.za)        |      "Lord, won't you buy me         |
 | Dept Microbiology           |          A Mer-ce-des Benz           |
 | University of Cape Town     |     My friends all have Porsches     |
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