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Genome Res. 13:1754-1764, 2003
©2003 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/03 $5.00
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Methods

Allele-Specific Holliday Junction Formation: A New Mechanism of Allelic Discrimination for SNP Scoring

Qinghong Yang1,3, Alla Lishanski1, Wendy Yang1, Sandra Hatcher2, Henrietta Seet2 and Jeffrey P. Gregg2

1 FreshGene, Inc., Concord, California 94520, USA 2 Department of Pathology, Molecular Pathology, UC Davis Medical Center, Sacramento, California 95817, USA

We report here a new mechanism for allelic discrimination—allele-specific Holliday Junction formation. The Holliday Junction (HJ) is a unique DNA structure that can be formed in a sequence-nonspecific manner by routine PCR. To cause the PCR-based HJ formation to occur in an allele-specific manner, the PCR primers are manipulated such that an extra mismatch next to a SNP of interest is introduced between a target and a reference amplicon and a GC-clamp is added. Based on this new mechanism, novel SNP genotyping methods were developed, including a homogeneous fluorescence polarization (FP) competition assay that requires neither labeled primers/probes nor expensive enzymes/substrates. Using this novel genotyping technology, we were able to convert >95% of SNP sequences into genotyping assays that work well under a universal set of assay conditions and achieved 100% accuracy in clinical samples.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.997703.

3 Corresponding author.
E-MAIL qinghong{at}freshgene.com; FAX (925) 681-4012.


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This article has been cited by other articles:


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 Clin. Chem.Home page
W. Yang, T. Yaoi, S. Huang, Q. Yang, S. Hatcher, H. Seet, and J. P. Gregg
Detecting the C282Y and H63D Mutations of the HFE Gene by Holliday Junction-Based Allele-Specific Genotyping Methods
Clin. Chem., January 1, 2005; 51(1): 210 - 213.
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