AP-PCR

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AP-PCR
Arbitrarily primed PCR: any form of PCR which uses primers of arbitrary sequence and which amplifies random, but discrete, sequences of chromosomal DNA; AP-PCR has been used for typing bacteria. PCR is initially carried out under low stringency, and the primers bind at various sites to each strand of heat-denatured chromosomal DNA; the binding of primers occurs at ‘best-fit’ sequences, and may include mismatches.
In some cases two primers bind with relative efficiency, on opposite strands, at locations separated by a few hundred bases. If synthesis can occur normally from these two primers, a further round of cycling under low-stringency conditions, followed by many cycles under high-stringency conditions, may produce copies of an amplicon delimited by the two best-fit sequences. In the phase of high-stringency cycling not all the primers will bind to their best-fit sequences – so that only a proportion of the amplicons produced under low-stringency conditions will be amplified in the high-stringency phase. The amplicons from a given sample are subjected to gel electrophoresis, and the stained bands of amplicons form the fingerprint. Strains are compared and classified on the basis of their fingerprints.
One advantage of this approach is that there is no need for prior knowledge of the genome sequence; there is no need to design specific primers, and any isolate is potentially typable. Results are generally reproducible under standardized conditions in a given laboratory, but comparable results will not necessarily be obtained in other laboratories unless the procedures are identical; reproducibility of results depends not only on the primer sequence but also e.g. on the particular type of polymerase used and on the initial procedure used for preparing the sample DNA. Some other named methods are based on the same principle – RAPD (random amplified polymorphic DNA) analysis and DAF (direct amplification fingerprinting). These methods may differ e.g. in the length of the primers used, annealing temperature for the primers, and the type of gel used for electrophoresis. The original AP-PCR employed
primers of 20–50 nucleotides, with an annealing temperature of about 40°C, and used an agarose gel. In RAPD the primers are typically 10–20 nt, the annealing temperature is ~36°C,
and products are separated in an agarose gel. DAF uses short primers (5–8 nt) at an annealing temperature of ~30°C; because there are many more, smaller products, electrophoresis
is carried out in a polyacrylamide gel, and silver staining is used to detect bands in the fingerprint