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A standard protocol for single nucleotide primer extension in the human genome using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
Author(s) -
Wise Cheryl A.,
Paris Maxime,
Morar Bharti,
Wang Wei,
Kalaydjieva Luba,
Bittles Alan H.
Publication year - 2003
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1038
Subject(s) - snp genotyping , genotyping , molecular inversion probe , single nucleotide polymorphism , primer extension , genetics , restriction fragment length polymorphism , population , computational biology , snp , snp array , chemistry , mass spectrometry , genotype , biology , chromatography , nucleotide , gene , medicine , environmental health
Abstract Analysis of single nucleotide polymorphisms (SNPs) has become an increasingly important area of research, with numerous applications in medical genetics, population genetics, forensic science, and agricultural biotechnology. Large‐scale SNP analyses require the development of methodologies that are economical, flexible, accurate and capable of automation. Primer extension in conjunction with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) is currently emerging as a potential method for high‐throughput SNP genotyping. We have evaluated a number of published primer extension methods and refined a simple and robust protocol to analyze human autosomal disease‐causing mutations and population genetic markers on the Y‐chromosome. Twelve different variant sites were examined, and homozygotes, heterozygotes and hemizygotes were accurately typed. A 100% concordance was observed between SNP genotypes obtained using the MALDI‐TOFMS technique and alternative genotyping methods, such as restriction fragment length polymorphism (RFLP) assays and denaturing high‐performance liquid chromatography (DHPLC). Since multiple polymorphisms can be detected in single reactions, the method provides a cost‐effective approach for SNP analysis. The protocol is also extremely flexible (able to accommodate new markers) and can be adapted to a number of platforms without the use of commercial kits. Copyright © 2003 John Wiley & Sons, Ltd.