Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection
Locked nucleic acids (LNAs) are synthetic nucleic acid analogs that bind to complementary target molecules (DNA, RNA or LNA) with very high affinity. At the same time, this binding affinity is decreased substantially when the hybrids thus formed contain even a single mismatched base pair. We have ex...
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Published in | Nucleic acids research Vol. 30; no. 17; p. e91 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
01.09.2002
Oxford Publishing Limited (England) |
Subjects | |
Online Access | Get full text |
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Summary: | Locked nucleic acids (LNAs) are synthetic nucleic acid analogs that bind to complementary target molecules (DNA, RNA or LNA) with very high affinity. At the same time, this binding affinity is decreased substantially when the hybrids thus formed contain even a single mismatched base pair. We have exploited these properties of LNA probes to develop a new method for single nucleotide polymorphism genotyping. In this method, very short (hexamer or heptamer) LNA probes are labeled with either rhodamine or hexachlorofluorescein (HEX), and their hybridization to target DNAs is followed by measuring the fluorescence polarization (FP) of the dyes. The formation of perfectly complementary double‐stranded hybrids gives rise to significant FP increases, whereas the presence of single mismatches results in very small or no changes of this parameter. Multiplexing of the assay can be achieved by using differentially labeled wild‐type and mutant specific probes in the same solution. The method is homogeneous, and because of the use of extremely short LNA probes, the generation of a universal set of genotyping reagents is possible. |
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Bibliography: | ark:/67375/HXZ-JZJSVRV8-6 To whom correspondence should be addressed at: Genospectra, Inc., 6519 Dumbarton Circle, Fremont, CA 94555, USA. Tel: +1 510 818 2633; Fax: +1 510 818 2610; Email: tnikiforov@genospectra.com istex:AE7B2135E42B50440ED9FE95E76BEDCAC35CCEDF Received May 1, 2002; Revised and Accepted July 2, 2002 local:gnf090 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0305-1048 1362-4962 1362-4962 |
DOI: | 10.1093/nar/gnf090 |