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.

High-throughput screening for evidence of association by using mass spectrometry genotyping on DNA pools

“…The average allele frequency SD of 0.018-0.021 we report is similar to the 0.021 reported for kinetic PCR (11), slightly greater than the 0.014 reported for primer extension-denaturing high-performance liquid chromatography (9), the 0.009-0.017 reported for fluorescent nucleotide primer extension-capillary electrophoresis (14,18), and the 0.011 reported for pyrosequencing (17), and less than the 0.038 reported for bioluminometric-primer extension (13). Further, mass spectrometry offers advantages in the potential for automation over several of these other methods.…”

“…Because the major experimental question is not the absolute allele frequencies, but whether there are allele frequency differences between cases and controls, a consistent under-or overestimate of pooled allele frequencies, if modest or correctable, would not preclude a method from use. Several methods for typing SNPs in pooled DNA, including mass spectrometry, have been described (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). These methods currently have varying suitability to a high-throughput setting.…”

Genetic suppression of the circadian Clock mutation by the melatonin biosynthesis pathway

“…We used a 3′ end primer mismatch method (50) whereby we introduced an artificial mismatch at the third position from the end of the 3′ end of the primer to obtain more significant allele detection than a mismatch at the final 3′ nucleotide alone (51). All reactions were prepared with the ABI SYBR Green PCR kit and carried out real-time PCR on an ABI 7700 with the following conditions: 95°C for 10 min followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. The primers were as follows: Aanat.…”

Sucrase-Isomaltase Gene Expression Is Inhibited by Mutant Hepatocyte Nuclear Factor (HNF)-1.ALPHA. and Mutant HNF-1.BETA. in Caco-2 Cells

“…Specific mRNA levels were quantitated by the real-time PCR method, using the DNA Engine Opticon2 System and DyNAmo HS SYBR Green qPCR kit (MJ Research). In HNF-1 ␣ T539fsdelC and HNF-1 ␤ R177X cells, transfected mutant or endogenous wild HNF-1 ␣ and wild HNF-1 ␤ gene expression was determined by the allele-specific primer method (20,21). Table 1.…”