Single-nucleotide polymorphism typing based on pyrosequencing chemistry and acryl-modified glass chip

A new method (termed as "chip-BAMPER" (bioluminometric assay coupled with modified primer extension reactions)) for single-nucleotide polymorphism (SNPs) genotyping was developed by pyrosequencing chemistry coupled with hydrogel chip immobilized with single-stranded target DNAs. The method is based on allele-specific extension reaction, which is switched by the base type in the 3' end of allele-specific primers. A genotype is determined by comparing the light intensity from a pair of gel pads, and the specificity is improved by introducing an artificially mismatched base at the third position upstream from the 3' end of the allele-specific primer. The big problem of chip-BAMPER is the ultra-high background of the detection mixture because apyrase could not be used. Here, we successfully prepared and used beaded apyrase, which can be removed from the detection mixture before sample typing, to decrease the high background due to adenosine 5'-triphosphate and inorganic pyrophosphate or sodium pyrophosphate decahydrate contamination. Unlike gel-based pyrosequencing, chip-BAMPER is highly sensitive because many bases are extended at a time in one extension reaction. Usually, less than 0.25 microL of PCR products can give a successful genotyping. To evaluate the method, four SNPs, OLR1-C15577T, OLR1-C14417G, PPARG-Pro12Ala, and PPARG-C2821T, were detected. To avoid the crosstalk between two adjacent spots in a gel-chip, mineral oil was dispensed to coat the gel-chip for physically separating two spots. It is shown that this new strategy of SNP typing based on the acryl-modified glass chip is highly sensitive, simple, inexpensive, and easy to be automated. It can be used for various applications of DNA analysis at a relative high-throughput.