Journal
ANALYTICAL BIOCHEMISTRY
Volume 398, Issue 1, Pages 83-92Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.10.042
Keywords
TthRNase HII; Molecular beacon; SNP genotyping; Fluorescence detection; dsDNA target
Funding
- National High Technology Research and Development Program of China [2006AA02Z108]
- National Basic Research Program of China [2007CB914504]
- National Science Foundation of China [30571012, 30700131]
- Shanghai Leading Academic Discipline Project [B205]
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Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation. SNPs are important markers that link sequence variations to phenotypic changes. Because of the importance of SNPs in the life and medical sciences, a great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. In this article, we describe a novel method for SNP genotyping based on differential fluorescence emission due to cleavage by Thermus thermophilus RNase Hill (TthRNase HII) of DNA heteroduplexes containing an SNP site-specific chimeric DNA-rN(1)-DNA molecular beacon (cMB). We constructed a loop sequence for a cMB that contains a single SNP-specific ribonucleotide at the central site. When the cMB probe is hybridized to a target double-stranded DNA (dsDNA), a perfect match of the cMB/DNA duplex permits efficient cleavage with TthRNase HII, whereas a mismatch in the duplex due to an SNP greatly reduces efficiency. Cleavage efficiency is measured by the incremental difference of fluorescence emission of the beacon. We show that the genotypes of 10 individuals at 12 SNP sites across a series of human leukocyte antigen (HLA) can be determined correctly with respect to conventional DNA sequencing. This novel TthRNase HII-based method offers a platform for easy and accurate SNP analysis. (C) 2009 Elsevier Inc. All rights reserved.
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