Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation
Mein CA., Barratt BJ., Dunn MG., Siegmund T., Smith AN., Esposito L., Nutland S., Stevens HE., Wilson AJ., Phillips MS., Jarvis N., Law S., De Arruda M., Todd JA.
Large-scale pharmacogenetics and complex disease association studies will require typing of thousands of single-nucleotide polymorphisms (SNPs) in thousands of individuals. Such projects would benefit from a genotyping system with accuracy > 99% and a failure rate < 5% on a simple, reliable, and flexible platform. However, such a system is not yet available for routine laboratory use. We have evaluated a modification of the previously reported Invader SNP-typing chemistry for use in a genotyping laboratory and tested its automation. The Invader technology uses a Flap Endonuclease for allele discrimination and a universal fluorescence resonance energy transfer (FRET) reporter system. Three hundred and eighty-four individuals were genotyped across a panel of 36 SNPs and one insertion/deletion polymorphism with Invader assays using PCR product as template, a total of 14,208 genotypes. An average failure rate of 2.3% was recorded, mostly associated with PCR failure, and the typing was 99.2% accurate when compared with genotypes generated with established techniques. An average signal-to-noise ratio (9:1) was obtained. The high degree of discrimination for single base changes, coupled with homogeneous format, has allowed us to deploy liquid handling robots in a 384-well microtitre place format and an automated end-point capture of fluorescent signal. Simple semiautomated data interpretation allows the generation of 25,000 genotypes per person per week, which is 10-fold greater than gel-based SNP typing and microsatellite typing in our laboratory. Savings on labor costs are considerable. We conclude that Invader chemistry using PCR products as template represents a useful technology for typing large numbers of SNPs rapidly and efficiently.