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Vol. 11, Issue 1, 163-169, January 2001

METHODS
High-Throughput SNP Genotyping by Allele-Specific PCR with Universal Energy-Transfer-Labeled Primers

Maxim V. Myakishev,1,3 Yuri Khripin,2 Stella Hu,1 and Dean H. Hamer1

1 Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA; 2 Intergen Discovery Products, Gaithersburg, Maryland 20877, USA

We have developed a new method for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The technique involves PCR amplification of genomic DNA with two tailed allele-specific primers that introduce priming sites for universal energy-transfer-labeled primers. The output of red and green light is conveniently scored using a fluorescence plate reader. The new method, which was validated on nine model SNPs, is well suited for high-throughput, automated genotyping because it requires only one reaction per SNP, it is performed in a single tube with no post-PCR handling, the same energy-transfer-labeled primers are used for all analyses, and the instrumentation is inexpensive. Possible applications include multiple-candidate gene analysis, genomewide scans, and medical diagnostics.

3 Corresponding author.

11:163-169 ©2001 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/01 $5.00
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