title: Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation.
abstract: 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 plate format and an automated end-point capture of fluorescent signal. Simple semiautomated data interpretation allows the generation of approximately 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.
authors.affiliation: Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2XY UK.
authors.forename: C A
authors.initials: CA
authors.lastname: Mein
authors.forename: B J
authors.initials: BJ
authors.lastname: Barratt
authors.forename: M G
authors.initials: MG
authors.lastname: Dunn
authors.forename: T
authors.initials: T
authors.lastname: Siegmund
authors.forename: A N
authors.initials: AN
authors.lastname: Smith
authors.forename: L
authors.initials: L
authors.lastname: Esposito
authors.forename: S
authors.initials: S
authors.lastname: Nutland
authors.forename: H E
authors.initials: HE
authors.lastname: Stevens
authors.forename: A J
authors.initials: AJ
authors.lastname: Wilson
authors.forename: M S
authors.initials: MS
authors.lastname: Phillips
authors.forename: N
authors.initials: N
authors.lastname: Jarvis
authors.forename: S
authors.initials: S
authors.lastname: Law
authors.forename: M
authors.initials: M
authors.lastname: de Arruda
authors.forename: J A
authors.initials: JA
authors.lastname: Todd
mesh_terms: D001483:Base Sequence; D003922:Diabetes Mellitus, Type 1 / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000737:chemistry; D045585:Flap Endonucleases; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000295:instrumentation / Q000379:methods; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic
chemical_list: D015345:Oligonucleotide Probes; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human