Evaluation of Single Nucleotide Polymorphism Typing with Invader on PCR Amplicons and Its Automation
- Charles A. Mein1,
- Bryan J. Barratt1,
- Michael G. Dunn1,
- Thorsten Siegmund1,
- Annabel N. Smith1,
- Laura Esposito1,
- Sarah Nutland1,
- Helen E. Stevens1,
- Amanda J. Wilson1,
- Michael S. Phillips2,
- Nancy Jarvis3,
- Scott Law3,
- Monika de Arruda3 and
- John A. Todd1
- 1Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2XY UK; 2Third Wave Technologies, Inc., Madison, Wisconsin 53719 USA; 3Merck Research Laboratories, West Point, Pennsylvania 19486 USA
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 ∼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.
Footnotes
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↵3 Corresponding author.
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E-MAIL john.todd{at}cimr.cam.ac.uk; FAX 44-2–334762-102.
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- Received November 8, 1999.
- Accepted January 18, 2000.