Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation - PubMed (original) (raw)
doi: 10.1101/gr.10.3.330.
B J Barratt, M G Dunn, T Siegmund, A N Smith, L Esposito, S Nutland, H E Stevens, A J Wilson, M S Phillips, N Jarvis, S Law, M de Arruda, J A Todd
Affiliations
- PMID: 10720574
- PMCID: PMC311429
- DOI: 10.1101/gr.10.3.330
Evaluation of single nucleotide polymorphism typing with invader on PCR amplicons and its automation
C A Mein et al. Genome Res. 2000 Mar.
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.
Figures
Figure 1
Data from 96 individuals for the marker CTLA4 g.49A>G plotted as gross signal from each allele (a) or as a ratio of A signal to G signal (b). Data were acquired from the 96-well format PCR-Invader assay.
Figure 2
Average ratios, ±1
s.d.
, from 384 individuals, of signal from common allele to rare allele for each genotype for each locus. Data were generated in a 96-well PCR-Invader format.
Figure 3
Average ratios, ±1
s.d.
, from 384 individuals, of signal from common allele to rare allele for each genotype for 9 loci in 384- and 96-well formats.
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