High-throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers

Abstract

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.

Figure 1

Structure of universal energy-transfer-labeled primers.

Figure 2

Assay scheme. The reaction steps for an A/G SNP are shown. (FL) Fluorescein, (SR) Sulforhodamine.

Figure 3

Fluorescence images of two SNP assays. A total of 21 samples of genomic DNA were analyzed for an A/T-SNP (rs363) and a T/G-SNP (MAOA). The multiwell PCR plate was photographed with an FMBIO II imager (Hitachi) equipped with red and green filters.

Figure 4

Fluorescence intensity measurements for four SNP assays. The 21 samples of genomic DNA (40 ng per reaction) were analyzed for an A/T SNP (RS363), a C/T SNP (CYP17), a C/G SNP (HTR2C), and a G/T SNP (MAOA). Each plate also contained three controls without DNA. The red and green fluorescence intensity was determined in a Victor II plate reader (Wallac).

Figure 5

Combination of two allele-specific reactions in a single tube increases specificity. Two genomic DNA samples with known homozygous genotypes (AA and TT) were analyzed at different dilutions for an A/T SNP (rs363). (Left) Each DNA sample was assayed twice: First, in a tube containing the allele-specific and ET-labeled primer for A, then in a tube containing an allele-specific and ET-primer for T. (Right) Each DNA sample was assayed in a single tube containing both A and T sets of primers. The red and green fluorescence intensity was determined in a Victor II plate reader (Wallac).

Figure 6

Genomic DNA dilution experiment. Three DNA samples of known genotypes (CC, GC, and GG) were assayed for a G/C SNP (HTR2C) at various concentrations (40-, 4-, 0.4-, and 0-ng/reaction). The red and green fluorescence intensity was determined in a Victor II plate reader (Wallac).