M. Grow, V. Phillips and R. Reynolds
Roche Molecular Systems, Alameda, CA
The AmpliTypeŽ PM+DQA1 Kit is a PCR-based assay for the determination of the allelic types of the LDLR, GYPA, HBGG, S7S8, GC and HLADQA1 loci. When a sample heterozygous for a given locus is typed, the assay is designed to yield probe dot intensities which are relatively comparable within that locus. This feature is especially important for the evaluation of sample mixtures. Selective probe signal reduction can occur if the heat denatured PCR product is allowed to cool prior to being dispensed into the assay tray during the preparation for the hybridization. As cooling occurs, the GC B and HLA DQA1 4.1 probe dots are the first to exhibit this phenomenon. We have demonstrated that this signal reduction is due to primer annealing and subsequent extension over the single-stranded sites to which the probes are targeted. Addition of EDTA to PCR product prior to the heat denaturation step eliminates the residual Taq Polymerase activity, thus eliminating selective probe signal reduction.
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