A standard protocol for single nucleotide primer extension in the human genome using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Faculty of Computing, Health and Science
School of Exercise, Biomedical and Health Science
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 methodolo- gies that are economical, flexible, accurate and capable of automation. Primer extension in conjunc- tion 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, heterozy- gotes 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 reac- tions, the method provides a cost-effective approach for SNP analysis. The protocol is also extre- mely flexible (able to accommodate new markers) and can be adapted to a number of platforms without the use of commercial kits.