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

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.

Figure 1

Schematic summary of the RFMP genotyping strategy. PCR was done with primers designed to introduce a typeIIS restriction endonuclease recognition sequence (FokI = BstF5I; GGATG) 9 bases ahead of the polymorphism site. The enzymatic cleavage of the products leads to excision of two oligonucleotide fragments (7 mer and 13 mer) containing the variation site, and then masses of the resulting oligonucleotide fragments were examined by MALDI-TOF MS. Cleavage sites of FokI and BstF5I, an isoschizomer for FokI, are indicated by filled and blank triangles, respectively, and restriction endonuclease recognition site and primers by shaded bar and shaded arrows, respectively. One-base gap replaced by the artificial sequences and potential SNP site are denoted by blank space and a bold italic letter, respectively.

Figure 2

Representative MALDI-TOF MS spectra in individual samples. Genotyping results for SNPs rs1801133; masses for a pair of 7 mer and 13 mer for C and T alleles were 2226.4/3975.6 and 2241.4/3959.6, respectivley and rs1801131; masses for a pair of 7 mer and 13 mer for A and C alleles were 2249.4/3955.6 and 2225.4/3980, respectivley. X- and y- axes represent relative ion abundance and mass to charge ratio, respectively.

Figure 3

Allele frequency measurements on pools for different polymorphisms. Expected allele frequencies are obtained by individual genotyping. The frequencies calculated from pool data were corrected for unequal allelic representation using at least eight mass spectra of heterozygotes. The error bars represent the standard deviation.