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Kinetic hairpin oligonucleotide blockers for selective amplification of rare mutations
Jia Y.1,2; Sanchez J.A.1; Wangh L.J.1
2014-08-01
Source PublicationScientific Reports
ISSN20452322
Volume4
Abstract

Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals.

DOI10.1038/srep05921
URLView the original
Indexed BySCI
Language英语
WOS Research AreaScience & Technology - Other Topics
WOS SubjectMultidisciplinary Sciences
WOS IDWOS:000339940900014
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Cited Times [WOS]:6   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionINSTITUTE OF MICROELECTRONICS
Corresponding AuthorWangh L.J.
Affiliation1.Department of Biology, Brandeis University, Waltham, MA 02453, USA
2.State Key Laboratory of Analog and Mixed-Signal VLSI, University of Macau, Macau, China
First Author AffilicationUniversity of Macau
Recommended Citation
GB/T 7714
Jia Y.,Sanchez J.A.,Wangh L.J.. Kinetic hairpin oligonucleotide blockers for selective amplification of rare mutations[J]. Scientific Reports,2014,4.
APA Jia Y.,Sanchez J.A.,&Wangh L.J..(2014).Kinetic hairpin oligonucleotide blockers for selective amplification of rare mutations.Scientific Reports,4.
MLA Jia Y.,et al."Kinetic hairpin oligonucleotide blockers for selective amplification of rare mutations".Scientific Reports 4(2014).
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