Accessing genetic variation: genotyping single nucleotide polymorphisms

There are intense continuing efforts to increase the throughput and accuracy, and reduce the costs, of methods for genotyping single nucleotide polymorphisms (SNPs). This is driven by the hope that SNPs can act as markers for identifying the genes that underlie multifactorial disorders. PCR, invented in the 1980s, allows the sensitivity and specificity required for genotyping SNPs in large diploid genomes. The PCR step is the principal limiting factor in the throughput of current SNP-genotyping assays. The large number of different SNP assays are based on a small number of reaction principles that have been combined with solid-phase or solution-based assay formats. Fluorescence is the most frequently used detection method. Allele-specific oligonucleotides as probes or PCR primers are used to achieve high throughput in homogeneous solution phase assays that are monitored in real time during PCR. Alternatively, large numbers of oligonucleotide probes are immobilized at high density on microarrays to allow parallel analysis of many SNPs. The most promising methods for accurate genotyping of SNPs involve nucleic-acid-modifying enzymes as genotyping tools. Frequently used enzymes are DNA polymerases, ligases and endonucleases. Assays based on primer extension catalysed by a DNA polymerase are robust and have been adapted to various assay formats and detection strategies. These include colorimetric detection in microtitre plates, fluorescence detection using DNA sequencers, mass spectrometric detection and microarray-based assays with fluorescence detection. Assays based on DNA ligation, or cleavage by FLAP endonucleases, have led to the development of SNP-genotyping methods in which a PCR amplification step is avoided. Instead, an enzymatic signal amplification scheme is used to obtain sufficient sensitivity. Future SNP assays could be based on PCR carried out in microcapillaries streamlined with one of the enzymatic detection principles, and the assays could be multiplexed by combinatorial fluorescent labels.