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Open Access Methodology article

A simple and accurate SNP scoring strategy based on typeIIS restriction endonuclease cleavage and matrix-assisted laser desorption/ionization mass spectrometry

Sun Pyo Hong1, Seung Il Ji1, Hwanseok Rhee1, Soo Kyeong Shin1, Sun Young Hwang1, Seung Hwan Lee2, Soong Deok Lee3, Heung-Bum Oh4, Wangdon Yoo1 and Soo-Ok Kim1*

Author Affiliations

1 Research & Development Center, GeneMatrix, Inc., Yongin, 446-913, South Korea

2 DNA Analysis Lab, Supreme Public Prosecutor's Office, Seoul 137-730, South Korea

3 Department of Forensic Medicine, Seoul National University College of Medicine, 28, Yongon-Dong, Chongno-Gu, Seoul, 110-799, South Korea

4 Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, 138-736, South Korea

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BMC Genomics 2008, 9:276  doi:10.1186/1471-2164-9-276

Published: 9 June 2008

Abstract

Background

We describe the development of a novel matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-based single nucleotide polymorphism (SNP) scoring strategy, termed Restriction Fragment Mass Polymorphism (RFMP) that is suitable for genotyping variations in a simple, accurate, and high-throughput manner. The assay is based on polymerase chain reaction (PCR) amplification and mass measurement of oligonucleotides containing a polymorphic base, to which a typeIIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products leads to excision of oligonucleotide fragments representing base variation of the polymorphic site whose masses were determined by MALDI-TOF MS.

Results

The assay represents an improvement over previous methods because it relies on the direct mass determination of PCR products rather than on an indirect analysis, where a base-extended or fluorescent report tag is interpreted. The RFMP strategy is simple and straightforward, requiring one restriction digestion reaction following target amplification in a single vessel. With this technology, genotypes are generated with a high call rate (99.6%) and high accuracy (99.8%) as determined by independent sequencing.

Conclusion

The simplicity, accuracy and amenability to high-throughput screening analysis should make the RFMP assay suitable for large-scale genotype association study as well as clinical genotyping in laboratories.