Microarray-based DNA resequencing using 3' blocked primers.

by: Jakub Sram, Steve S S Sommer, Qiang Liu

Anal Biochem (4 November 2007)

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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 potential for accurate and high-throughput microarray-based resequencing.

@article{citeulike:2048823, 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 potential for accurate and high-throughput microarray-based resequencing.}, address = {Department of Molecular Genetics, City of Hope National Medical Center, Duarte, CA 91010, USA.}, author = {Sram, Jakub and Sommer, Steve S S. and Liu, Qiang }, citeulike-article-id = {2048823}, doi = {10.1016/j.ab.2007.10.044}, issn = {0003-2697}, journal = {Anal Biochem}, month = {November}, posted-at = {2008-05-12 16:42:31}, priority = {2}, title = {Microarray-based DNA resequencing using 3' blocked primers.}, url = {http://dx.doi.org/10.1016/j.ab.2007.10.044}, year = {2007} }

RIS record

TY - JOUR ID - citeulike:2048823 L3 - citeulike-article-id:2048823 TI - Microarray-based DNA resequencing using 3' blocked primers. JF - Anal Biochem AD - Department of Molecular Genetics, City of Hope National Medical Center, Duarte, CA 91010, USA. SN - 0003-2697 N2 - 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. AU - Sram, Jakub AU - Sommer, Steve S AU - Liu, Qiang PY - 2007/11/04/ UR - http://dx.doi.org/10.1016/j.ab.2007.10.044 ER -

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