High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR - PubMed (original) (raw)

High-throughput SNP allele-frequency determination in pooled DNA samples by kinetic PCR

S Germer et al. Genome Res. 2000 Feb.

Abstract

We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r(2) = 0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.

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Figures

Figure 1

Figure 1

The basis of allele frequency measurement using kinetic PCR. Shown are amplification growth curves of PCR reactions performed for the ApoB71 polymorphism. A sample was constructed from two DNAs each homozygous for the different alleles of the ApoB71 SNP and contains 5% of allele 1. Equal aliquots of the pool (20 ng of DNA each) were put into PCRs containing either of the two allele-specific primer sets. Four replicate reactions were performed with each primer set (eight PCRs total). The relative allele frequency is determined on the basis of the Δ_Ct_ using equation 1 (see text and Fig. 2).

Figure 2

Figure 2

The relationship between Δ_Ct_ and allele frequency. The solid center line is a plot of equation 1 from the text. The flanking solid lines represent the expected uncertainty (1

s.d.

) in estimating the allele frequency based on sampling error alone (sample size = 1000). The broken lines represent the combined uncertainty of sampling and measurement error. The measurement error is based on an average error seen amongst the measurements taken in this paper and is that expected after averaging four replicate measurements. The insets compare the impact of measurement error at the middle and at the upper extreme of allele frequencies (the lower extreme should mirror exactly the upper).

Figure 3

Figure 3

The accuracy of allele frequency measurement by kinetic PCR. Shown is a scatter-plot of all the measurements of allele frequency made in Tables 1, 2, and 3 comparing the known frequencies (determined by DNA concentration for Table 1 and by individual genotyping and allele counting for Tables 1 and 2) with the measured frequencies. The error bars represent one

s.d.

in the measurement. The diagonal line is that expected for complete concordance between known and measured values.

Figure 4

Figure 4

The impact of measurement error for three SNP assays. (A) Plotted as the solid line is the expected sampling error for this SNP given an allele frequency of 10% (see text) for sample sizes up to 1000. The upper broken line is the estimated combined sampling and measurement error for this assay based on Table 3 and using the average of four measurements. This measurement error alone is the lower broken line. (B) The same as A for the human CST5 locus (Table 2). (C) The same as A and B for the mouse REN1 SNP (Table 3).

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