High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers.
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.
Myakishev
M V
MV
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. myakishm@mail.nih.gov
Khripin
Y
Y
Hu
S
S
Hamer
D H
DH
D000483:Alleles; D017931:DNA Primers / Q000138:chemical synthesis / Q000235:genetics*; D004735:Energy Transfer / Q000235:genetics*; D019793:Fluorescein; D005456:Fluorescent Dyes; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity; D014966:Xanthenes
D017931:DNA Primers; D005456:Fluorescent Dyes; D014966:Xanthenes; C034657:Texas red; D019793:Fluorescein