DNA analysis by fluorescence quenching detection

Abstract

The analysis of human genetic variations such as single nucleotide polymorphisms (SNPs) has great applications in genome-wide association studies of complex genetic traits. We have developed an SNP genotyping method based on the primer extension assay with fluorescence quenching as the detection. The template-directed dye-terminator incorporation with fluorescence quenching detection (FQ-TDI) assay is based on the observation that the intensity of fluorescent dye R110- and R6G-labeled acycloterminators is universally quenched once they are incorporated onto a DNA oligonucleotide primer. By comparing the rate of fluorescence quenching of the two allelic dyes in real time, we have extended this method for allele frequency estimation of SNPs in pooled DNA samples. The kinetic FQ-TDI assay is highly accurate and reproducible both in genotyping and in allele frequency estimation. Allele frequencies estimated by the kinetic FQ-TDI assay correlated well with known allele frequencies, with an r(2) value of 0.993. Applying this strategy to large-scale studies will greatly reduce the time and cost for genotyping hundreds and thousands of SNP markers between affected and control populations.

Figure 1.

The quenching patterns for four dye-labeled acycloterminators, R110, R6G, Tamra, and Texas Red. The quenching pattern for R110-acycloterminators (black bars) was derived from results of over 667 SNP primers. Red, green, and yellow bars represent the quenching patterns of R6G (258 SNP primers), Tamra (667 SNP primers), and Texas Red (50 SNP primers) acycloterminators, respectively. The x-axis is the percentage of quenching, which are grouped in 10% intervals from  − 10%–0% to 90%–100%. The y-axis is the percentage of SNP primers in each of the quenching fractions.

Figure 2.

The real-time fluorescence intensity profiles of four representative samples tested for SNP marker rs154162 during thermal cycling of the primer extension step of the TDI assay. The fluorescence readings were at the emission maxima for R110-G and R6G-A acycloterminators using multicomponent analysis to subtract contributions of fluorescence from the other dye at those wavelengths based on the pure spectra of R110 and R6G. (•) R110 fluorescence; (○) R6G fluorescence. The intensity profiles of (A) a G/G homozygous sample, (B) an A/A homozygous sample, (C) a G/A heterozygous sample, and (D) negative control.

Figure 3.

The basis of determining allele frequency for pooled DNA samples. The top panel is a normalized real-time quenching curve for a heterozygous sample of rs922365. The linear regression of the first eight cycles is shown in the inset. The bottom panel is the quenching curve for a mixture containing 75% A allele.

Figure 4.

The plot of slope ratio vs. allele frequency using data in Table 1. The scatterplot is the slope ratio vs. known allele frequency. The curve fitting is with the hyperbolic equation y = a/(1 + bX) with a and b being 1.02 (P < 0.0001) and 0.66 (P < 0.0001), respectively, which agrees well with predicted values of 1 and 0.67.

Figure 5.

Allele frequency measurements in pools (y-axis) vs. allele frequency obtained by genotyping individuals (x-axis). A linear relation was observed with r² = 0.993, slope = 1.002 (P < 0.0001).