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Open Access Research article

Robust SNP genotyping by multiplex PCR and arrayed primer extension

Mohua Podder12, Jian Ruan1, Ben W Tripp1, Zane E Chu14 and Scott J Tebbutt13*

Author Affiliations

1 The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital, University of British Columbia, Vancouver, BC, V6Z 1Y6, Canada

2 Department of Statistics, University of British Columbia, Vancouver, BC, V6T 1Z2, Canada

3 Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, BC, V6Z 1Y6, Canada

4 Current Address: Division of Engineering Science, University of Toronto, Toronto, ON, M5S 2E4, Canada

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BMC Medical Genomics 2008, 1:5  doi:10.1186/1755-8794-1-5

Published: 31 January 2008

Abstract

Background

Arrayed primer extension (APEX) is a microarray-based rapid minisequencing methodology that may have utility in 'personalized medicine' applications that involve genetic diagnostics of single nucleotide polymorphisms (SNPs). However, to date there have been few reports that objectively evaluate the assay completion rate, call rate and accuracy of APEX. We have further developed robust assay design, chemistry and analysis methodologies, and have sought to determine how effective APEX is in comparison to leading 'gold-standard' genotyping platforms. Our methods have been tested against industry-leading technologies in two blinded experiments based on Coriell DNA samples and SNP genotype data from the International HapMap Project.

Results

In the first experiment, we genotyped 50 SNPs across the entire 270 HapMap Coriell DNA sample set. For each Coriell sample, DNA template was amplified in a total of 7 multiplex PCRs prior to genotyping. We obtained good results for 41 of the SNPs, with 99.8% genotype concordance with HapMap data, at an automated call rate of 94.9% (not including the 9 failed SNPs). In the second experiment, involving modifications to the initial DNA amplification so that a single 50-plex PCR could be achieved, genotyping of the same 50 SNPs across each of 49 randomly chosen Coriell DNA samples allowed extremely robust 50-plex genotyping from as little as 5 ng of DNA, with 100% assay completion rate, 100% call rate and >99.9% accuracy.

Conclusion

We have shown our methods to be effective for robust multiplex SNP genotyping using APEX, with 100% call rate and >99.9% accuracy. We believe that such methodology may be useful in future point-of-care clinical diagnostic applications where accuracy and call rate are both paramount.