Multiplex universal genotyping using a modified ARMS-PCR protocol.

DNA mutations underlie the pathology of inherited disease. The spectrum of genetic changes resulting in disease is diverse, often encompassing both point (single-nucleotide) and length (large deletions) mutations: cystic fibrosis (2) and the muscular dystrophies (10) are classic examples. Similarly, mutational events implicated in carcinogenesis include both single nucleotide substitutions and multi-base pair deletions (1). In contrast to disease-causing mutations, genetic polymorphisms occur frequently in human populations. SNPs, occurring in the human genome with an average frequency greater than 1:1000 bp, account for most of the sequence variation between individuals (5). Their high frequency combined with their relatively even distribution across the entire genome makes them ideal markers for studies of genetic linkage and disease susceptibility. Disease association studies explore the hypothesis that certain allelic variants of candidate genes, arising either from SNPs or length polymorphisms, contribute to the overall risk or severity of disease (7). The mass identification of new polymorphisms by the Human Genome Project and the private sector has renewed interest in studying the complex genetics of multifactorial diseases. At the same time, the sheer abundance of polymorphisms, even within single genes, imposes methodological hurdles that are hard to overcome, resulting in mounting criticism of such studies (9). In the absence of data pertaining to the phenotypic impact of individual polymorphisms, the definition of polymorphic haplotypes through linkage analysis offers one way of addressing this problem (7). Therefore, linkage and disease association studies, in addition to clinical diagnostics, would benefit from the development of a universal genotyping tool. Despite the recent development of several high-throughput genotyping technologies, there is as yet no single assay that allows the simultaneous analysis of both point and length polymorphisms. The amplification refractory mutation system (ARMS-PCR) (4), also known as PCR using sequence-specific primers (PCR-SSP), is an established platform for genotyping SNPs. This technique exploits the relative inability of Taq DNA polymerase to extend primers mismatched at their 3′-end as well as its intrinsic lack of 3′→5′ exonuclease activity. As SNPs are virtually always biallelic, ARMS-PCR determines the allelic status of any SNP using two duplex reactions: each one employs either allele-specific primer paired with a common companion primer, as well as a primer pair amplifying an irrelevant locus of suitable, fixed length. The latter reaction should proceed independently of the allelespecific reaction; thus, it serves as an internal control. Here I describe a simple modification of ARMS-PCR that extends its applicability to the study of length polymorphisms. The essence of this modification lies in using the internal control primer pair to amplify a length polymorphism of interest. Since this reaction remains allele nonspecific, it still doubles as an internal control. I have recently used this modified ARMS-PCR protocol to study linkage disequilibrium in the human IL1 gene cluster (12) and to identify polymorphisms associated with chronic graft nephropathy (11). Assay development is essentially a three-step process. Initially, primers to amplify the length polymorphism of interest are designed, and all length variants are characterized. In the example illustrated here, a primer pair (5′-TGGCCTTGTTCATTTTCCCTGC-3′ and 5′-TCATCTTCCTGGTCTGCAGGTA-3′) was designed to amplify a minisatellite occurring within intron 2 of the human IL1RN gene (6). This particular hexaallelic polymorphism involves a tandemly repeated 86-bp sequence (8) and has been associated with a diverse spectrum of pathologies, ranging from osteoporosis to cardiovascular disease (14). Subsequently, allele-specific primers are designed for the SNP of interest (see Reference 3 for guidelines), along with a common companion primer. The size of the allele-specific product is crucial, as this should be resolvable from the length polymorphism DRUG DISCOVERY AND GENOMIC TECHNOLOGIES