Quantitative analysis of human DNA sequences by PCR and solid-phase minisequencing

Reliable quantification by PCR requires careful experimental design and conditions, often involving sampling of the PCR reactions at different time points or amplifying multiple dilutions of a standard DNA. We describe here an accurate, quantitative and easily automatizable solid-phase method based on competitive PCR. The PCR products are analyzed by solid-phase mini-sequencing after capture of biotinylated PCR products in streptavidin-coated microtiter wells and single-nucleotide extension of a specific detection primer by a radioactively labelled nucleotide. The results are expressed as numeric cpm-values, and the incorporated label expresses the relative amount of sequence variants in the original template mixture. We have applied the method to determination of allele frequencies in pooled DNA samples, of mitochondrial heteroplasmy, of gene copy numbers, and to forensic DNA analysis.