Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry

The high frequency of single nucleotide polymorphisms (SNPs) in the human g enome makes them a valuable source of genetic markers for identity testing, genome mapping, and medical diagnostics. Conventional technologies for det ecting SNPs are laborious and time-consuming, often prohibiting large-scale analysis. A rapid, accurate, and cost-effective method is needed to meet t he demands of a high-throughput DNA assay. We demonstrate here that analysi s of these genetic markers can now be performed routinely in a rapid, autom ated, and high-throughput fashion using time-of-flight mass spectrometry an d a primer extension assay with a novel cleavable primer. SNP genotyping by mass spectrometry involves detection of single-base extension products of a primer immediately adjacent to the SNP site. Measurement of the mass diff erence between the SNP primer and the extension peak reveals which nucleoti de is present at the polymorphic site. The primer is designed such that its extension products can be purified and chemically released from the primer in an automated format. The reduction in size of the products as a result of this chemical cleavage allows more accurate identification of the polymo rphic base, especially in samples from a heterozygotic population. All six possible heterozygotes are resolved unambiguously, including an AVT heteroz ygote with extension products differing by only 9 Da. Multiplex SNP determi nation is demonstrated by simultaneously probing multiple SNP sites from a single polymerase chain reaction (PCR) product as well as from multiplexed PCR amplicons. Samples are processed in parallel on a robotic workstation, and analyzed serially in an automated mass spectrometer with analysis times of only a few seconds per sample, making it possible to process thousands of samples per day.