Microarray-based DNA resequencing using 3′ blocked primers

To exceed the throughput and accuracy of conventional sequencing technologies, we tested a method (pyrophosphorolysis-activated polymerization [PAP]) of nucleic acid amplification that uses 3′ blocked primers (P∗s). As proof-of-principle, we resequenced a 20-bp region of the factor IX gene with a microarray of P∗s. P∗s discriminate 3′ end mismatches with ultra-high specificity as well as mismatches along their lengths with high specificity. We correctly identified two wild-type samples as well as all mismatches, including three single-base substitutions, one microdeletion, one microinsertion, and one heterozygous mutation. Despite limitations in the primer purity, the signal/noise ratio between the matched and mismatched P∗s sometimes exceeded 1000. Thus, PAP resequencing shows great potential for accurate and high-throughput microarray-based resequencing.