Microarray-based DNA resequencing using 3' blocked primers.

ABSTRACT: 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 poten

SUBMITTER: Sram J 

PROVIDER: S-EPMC2292398 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature