CHARACTERIZATION OF POLYMERASE CHAIN-REACTION AMPLIFICATION OF SPECIFIC ALLELES

Under certain conditions, polymerase chain reaction (PCR) can be used to differentially amplify one allele over another. To characterize the phenomenon, we have made a series of PCR primers and determined whether differential amplification could be detected after agarose gel electrophoresis. Two allele pairs were examined; one pair represents a transversion and one pair represents a transition. The following conclusions emerge: (i) when the 3′ or the 3′ penultimate base of the oligonucleotide mismatched an allele, no amplification product could be detected; (ii) when the mismatches were 3 and 4 bases from the 3′ end of the primer, differential amplification was still observed, but only at certain concentrations of magnesium chloride; (iii) the mismatched allele can be detected in the presence of a 40-fold excess of the matched allele; (iv) primers as short as 13 nucleotides were effective; and (v) the specificity of the amplification could be overwhelmed by greatly increasing the concentration of target DNA. © 1990.