Quantitative detection of single nucleotide polymorphisms for a pooled sample by a bioluminometric assay coupled with modified primer extension reactions (BAMPER)

Abstract

A new method for SNP analysis based on the detection of pyrophosphate (PPi) is demonstrated, which is capable of detecting small allele frequency differences between two DNA pools for genetic association studies other than SNP typing. The method is based on specific primer extension reactions coupled with PPi detection. As the specificity of the primer-directed extension is not enough for quantitative SNP analysis, artificial mismatched bases are introduced into the 3'-terminal regions of the specific primers as a way of improving the switching characteristics of the primer extension reactions. The best position in the primer for such artificial mismatched bases is the third position from the primer 3'-terminus. Contamination with endogenous PPi, which produces a large background signal level in SNP analysis, was removed using PPase to degrade the PPi during the sample preparation process. It is possible to accurately and quantitatively analyze SNPs using a set of primers that correspond to the wild-type and mutant DNA segments. The termini of these primers are at the mutation positions. Various types of SNPs were successfully analyzed. It was possible to very accurately determine SNPs with frequencies as low 0.02. It is very reproducible and the allele frequency difference can be determined. It is accurate enough to detect meaningful genetic differences among pooled DNA samples. The method is sensitive enough to detect 14 amol ssM13 DNA. The proposed method seems very promising in terms of realizing a cost-effective, large-scale human genetic testing system.

Figure 1

The principles of allele frequency assay using PPi detection coupled with strand extension of two primers specific to the allele. Only matched primers can be extended to produce PPi. The PPi is then used to emit bioluminescence.

Figure 2

A mismatch base artificially introduced into primers at the third position from the 3′-terminus improved the switching characteristics of primer extension reactions. To detect the mutant or wild-type DNAs, two extension reactions are carried out and the emissions were compared to determine the heterozygosity or allele frequency.

Figure 3

Comparison of primer extensions for wild-type and mutant targets. In (A) there is no artificial mismatch base in the primer; while in (B)–(D) an artificial mismatch base has been placed at the second, third and fourth positions from the 3′-terminus of the primer, respectively. The sequences of primers A, C, T and G were the same except for the base at the 3′-terminus. The concentrations of template, primers and each dNTP were 25 nM, 50 nM and 50 µM, respectively. The injection volume of the sample was 1 µl, which includes 25 fmol template. The intensities shown were normalized so that the maximum value in each figure is 100.

Figure 4

Signal intensity change from decomposition of dNTPs for 2 min at various temperatures. The concentration of each dNTP was 20 nM.

Figure 5

Signal intensity of 50 nM template at different concentrations of dNTPs. In the extension step the concentrations of template (P53Wt) and of primer were 50 and 100 nM, respectively. One microliter was injected for the determination. The concentration of each dNTP was the same.

Figure 6

Comparison of signal intensities obtained with the prototype instrument in two different modes, pyrosequencing and BAMPER. The experimental conditions were the same, except that apyrase was not used in BAMPER. The sample was 0.5 fmol ssM13. BG indicates the background signal level.

Figure 7

A bioluminescence assay with modified primer extension reactions for genotyping. Targets containing A (P53Wt) and T (P53Mut) at the polymorphic site were used. For homo-templates it was only possible to extend one primer. However, both primers extended for a hetero-template.

Figure 8

Results of using BAMPER to type 16 SNPs. The intensity was normalized to the total intensity. The details of each SNP, including the corresponding gene name, mutation point and specific primers are given in Table 2.

Figure 9

Comparison of sampling errors (black and purple lines) and measurement errors (error bars) at different allele frequencies. Sample sizes were 100 for the black line and 1000 for the purple line, respectively. The green line was drawn by the regression equation obtained using the observed allele frequency and the known allele frequency. The error bar represents the measurement error described in Table 3.