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Open Access Highly Accessed Research article

High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification

A Fawad Faruqi1, Seiyu Hosono1, Mark D Driscoll1, Frank B Dean1, Osama Alsmadi1, Rajanikanta Bandaru1, Gyanendra Kumar1, Brian Grimwade1, Qiuling Zong1, Zhenyu Sun1, Yuefen Du1, Stephen Kingsmore1, Tim Knott2 and Roger S Lasken1*

Author affiliations

1 Molecular Staging Inc., 300 George St., Suite 701, New Haven, CT 06511, USA

2 Amersham Pharmacia Biotech, Amersham Laboratories, White Lion Road, Amersham, Buckinghamshire, HP7 9LL, England

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Citation and License

BMC Genomics 2001, 2:4  doi:10.1186/1471-2164-2-4

Published: 1 August 2001

Abstract

Background

Single nucleotide polymorphisms (SNPs) are the foundation of powerful complex trait and pharmacogenomic analyses. The availability of large SNP databases, however, has emphasized a need for inexpensive SNP genotyping methods of commensurate simplicity, robustness, and scalability.

We describe a solution-based, microtiter plate method for SNP genotyping of human genomic DNA. The method is based upon allele discrimination by ligation of open circle probes followed by rolling circle amplification of the signal using fluorescent primers. Only the probe with a 3' base complementary to the SNP is circularized by ligation.

Results

SNP scoring by ligation was optimized to a 100,000 fold discrimination against probe mismatched to the SNP. The assay was used to genotype 10 SNPs from a set of 192 genomic DNA samples in a high-throughput format. Assay directly from genomic DNA eliminates the need to preamplify the target as done for many other genotyping methods. The sensitivity of the assay was demonstrated by genotyping from 1 ng of genomic DNA. We demonstrate that the assay can detect a single molecule of the circularized probe.

Conclusions

Compatibility with homogeneous formats and the ability to assay small amounts of genomic DNA meets the exacting requirements of automated, high-throughput SNP scoring.