||Single nucleotide polymorphism ( SNP ) is the most frequent type of genetic variation in human. Certain SNPs are related to cancers, drug metabolism, and genetic disorders. A wide variety of techniques have been developed to analyze SNPs, including DNA microarray, pyrosequencing, and Sanger sequencing.
This manuscript is based on upon Bioluminometric Assay Coupled With Modified Primer Extension Reactions ( BAMPER ). When a specifically designed primer is fully complementary to the template containing SNP site, the DNA polymerase can incorporate the deoxynucleotides to extend the primer. One PPi molecule is released whenever a deoxynucleotide is attached to the growing primer. PPi is further converted to green-yellow bioluminescence (562 nm) by ATP sulfurylase and firefly luciferase. However, when the primer is not fully complementary to the template, the bioluminescence intensity is greatly decreased. The SNP genotype can be identified by comparing bioluminescence intensity when a pair of SNP primers are used.
A plasmid（pET-23a (＋)）containing D-amino acid oxidase gene was used as model analyte. When artificial mismatch primer（AMP）was applied, the bioluminescence ratio was 1.0 ± 0.07 (n=3) for heterozygous genotype. For homozygous genotypes, the ratios were 5.6 ± 1.8 (mutant, n = 3) and 4.5 ± 1.1 (wild type, n = 3). Considering three times of the standard deviation of the heterozygous, result with bioluminescence ratio greater than 1.21 can be called as homozygous. We also attempted to clean up the background PPi in the deoxynucleotides, nonetheless, the background removal did not facilitate the SNP identification.
In order to develop microfluidic SNP analysis device, we tested the possibility of using immobilized enzymes in BAMPER. The Immunodyne® ABC Membrane was selected as support and three different combinations of enzyme concentrations were tested. 0.71 mg/mL firefly luciferase and 0.14 mg/mL ATP sulfurylase were found optimal.