title: A standard protocol for single nucleotide primer extension in the human genome using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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.
authors.affiliation: Centre for Human Genetics, Edith Cowan University, Perth, Australia. c.wise@ecu.edu.au
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Wise
authors.forename: Maxime
authors.initials: M
authors.lastname: Paris
authors.forename: Bharti
authors.initials: B
authors.lastname: Morar
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Luba
authors.initials: L
authors.lastname: Kalaydjieva
authors.forename: Alan H
authors.initials: AH
authors.lastname: Bittles
mesh_terms: D041322:Chromosomes, Human, Y / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers / Q000235:genetics* / Q000378:metabolism*; D005820:Genetic Testing / Q000379:methods; D015894:Genome, Human; D005838:Genotype; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D017931:DNA Primers