Multiplex Universal Genotyping Using a Modified ARMS-PCR Protocol

DNA mutations underlie the pathol-ogy of inherited disease. The spectrumof genetic changes resulting in diseaseis diverse, often encompassing bothpoint (single-nucleotide) and length(large deletions) mutations: cystic fi-brosis (2) and the muscular dystrophies(10) are classic examples. Similarly,mutational events implicated in car-cinogenesis include both single nu-cleotide substitutions and multi-basepair deletions (1).In contrast to disease-causing muta-tions, genetic polymorphisms occur fre-quently in human populations. SNPs,occurring in the human genome with anaverage frequency greater than 1:1000bp, account for most of the sequencevariation between individuals (5). Theirhigh frequency combined with their rel-atively even distribution across the en-tire genome makes them ideal markersfor studies of genetic linkage and dis-ease susceptibility. Disease associationstudies explore the hypothesis that cer-tain allelic variants of candidate genes,arising either from SNPs or lengthpolymorphisms, contribute to the over-all risk or severity of disease (7).The mass identification of new poly-morphisms by the Human GenomeProject and the private sector has re-newed interest in studying the complexgenetics of multifactorial diseases. Atthe same time, the sheer abundance ofpolymorphisms, even within singlegenes, imposes methodological hurdlesthat are hard to overcome, resulting inmounting criticism of such studies (9).In the absence of data pertaining to thephenotypic impact of individual poly-morphisms, the definition of polymor-phic haplotypes through linkage analy-sis offers one way of addressing thisproblem (7). Therefore, linkage anddisease association studies, in additionto clinical diagnostics, would benefitfrom the development of a universalgenotyping tool. Despite the recent de-velopment of several high-throughputgenotyping technologies, there is as yetno single assay that allows the simulta-neous analysis of both point and lengthpolymorphisms.The amplification refractory muta-tion system (ARMS-PCR) (4), alsoknown as PCR using sequence-specificprimers (PCR-SSP), is an establishedplatform for genotyping SNPs. Thistechnique exploits the relative inabilityof