POST-AMPLIFICATION PRIMER EXTENSION OF HEAT-DENATURED AMPLITYPE(R) PCR PRODUCTS - EFFECTS ON TYPING RESULTS

Alleles of the HLA DQA1, LDLR, GYPA, HGBB, D7S8 and GC loci, which are amplified using the AmpliType(R) PM PCR Reaction Mix and Primer Set, can be detected using sequence-specific oligonucleotide probes immobil ized on a nylon membrane strip. Using reagents supplied in AmpliType P CR Amplification and Typing Kits, patterns of blue dots corresponding to particular alleles are visualized on the DNA probe strips. Frequent ly the correct interpretation of typing results is dependent not only on the presence of probe signals but also on their relative intensitie s. The relative probe signal intensities obtained from an undegraded D NA sample extracted from a single individual will be different from th ose obtained from degraded DNA and from samples containing DNA from mo re than one source. Because probe signal intensity is an essential con sideration for interpretation, factors that can influence it need to b e identified. Clearly, the time and temperature of the assay steps and the salt concentration in the typing solutions can affect probe signa l intensity. Also, if heat-denatured PCR products are allowed to cool for several minutes, the strands will reanneal and become unavailable for binding to the probes immobilized on the strips. However, the sele ctive loss of GC B and HLA DQA1 4.1 probe signals observed after short er cooling times cannot be explained by these factors. We demonstrate that following heat denaturation of PM PCR products there is sufficien t residual Tag DNA polymerase activity to extend primers as the soluti on cools and that this primer extension occurs at a more rapid rate th an PCR product reannealing. Primer extension across probe binding site s will prevent hybridization of the PCR product to complementary probe s on the strip. The extent of signal reduction is dependent on the pos ition of the probe binding site relative to the 3' ends of the primers and on the strand to which the probe is complementary. We recommend a simple modification to the AmpliType typing protocol to ensure all pr obe binding sites will be available for hybridization to PM and HLA DQ A1 DNA probe strips.