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Vol. 10, Issue 3, 330-343, March 2000

METHODS
Evaluation of Single Nucleotide Polymorphism Typing with Invader on PCR Amplicons and Its Automation

Charles A. Mein,1 Bryan J. Barratt,1 Michael G. Dunn,1 Thorsten Siegmund,1 Annabel N. Smith,1 Laura Esposito,1 Sarah Nutland,1 Helen E. Stevens,1 Amanda J. Wilson,1 Michael S. Phillips,2 Nancy Jarvis,3 Scott Law,3 Monika de Arruda,3 and John A. Todd1

1 Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2XY UK; 2 Third Wave Technologies, Inc., Madison, Wisconsin 53719 USA; 3 Merck Research Laboratories, West Point, Pennsylvania 19486 USA

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 ~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.

3 Corresponding author.

10:330-343 ©2000 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/00 $5.00
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