Electrospray Ionization Mass Spectrometry-Based Genotyping:  An Approach for Identification of Single Nucleotide Polymorphisms

The high frequency of single nucleotide polymorphisms (SNPs) in the human genome makes them ideal genetic markers for mapping, diagnosing disease-related alleles, and identifying SNPs that contribute to drug response differences between individuals. Here we report a novel assay utilizing a single nucleotide primer extension (SNuPE) and electrospray ionization mass spectrometry (ESI-MS) detection for the analysis of SNPs. In contrast to most SNuPE genotyping technologies that detect the extended primer product, the novel Survivor assay detects the unreacted dideoxynucleotides (ddNTPs) remaining or surviving in solution following a SNuPE. This assay involves a simple analysis of the same four ddNTP analytes, regardless of the SNP being investigated, and either single or double-stranded DNA can be used to genotype a SNP, without any labeling requirements of the ddNTPs or oligonucleotide primers. We have tested and blindly validated the Survivor assay by genotyping the C/T SNP at −857 of the human TNFα promoter gene. The results obtained are in agreement with the control sequencing data. The results demonstrate that the homogeneous Survivor assay with ESI-MS detection offers advantages in simplicity, accuracy, specificity, and sensitivity. Additional advantages of the method include enhanced hybridization efficiencies in this solution-phase assay and the elimination of immobilized primers for the isolation of single-stranded DNA. With a one-well reaction and an automation platform being developed, the Survivor assay provides a powerful new tool for large-scale SNP analysis and screening.