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.
Sram
Jakub
J
Department of Molecular Genetics, City of Hope National Medical Center, Duarte, CA 91010, USA.
Sommer
Steve S
SS
Liu
Qiang
Q
D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers