Abstract
BackgroundWe 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.ResultsThe 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.ConclusionThe 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.
Highlights
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 RFMP assay for SNP scoring utilizing mass difference of oligonucleotides requires the simple steps of single polymerase chain reaction (PCR) amplification and restriction enzyme digestion, and is amenable to high-throughput system
The assay represents an improvement over previous methods in reliance on the direct mass determination of PCR products rather than on an indirect analysis, where a baseextended or fluorescent report tag is interpreted, both DNA strands being analyzed in parallel, and the ensured specific target amplification simultaneously with mass analysis, providing an additional level of assay precision
Summary
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. Due to the complexity of many common, chronic diseases and quantitative traits and the confounding effects of disease heterogeneity, gene-gene interaction, and gene-environment interaction, a large number of the polymorphisms must be surveyed in numerous individuals These progresses highlight the need for rapid, accurate, and efficient methods that permit high throughput genotyping. Procedures have been widely used that use PCR products as templates to which oligonucleotide primers are hybridized, base-extended and analyzed by mass spectrometry. These assays can be useful, but they fail to employ one of key advantages of mass spectrometry that the analysis of PCR products can be direct. Only a small number of polymorphisms will alter known restriction sites, and the design of amplification primers to create restriction sites in connection with one allele is not straightforward in most cases, reducing the usefulness of this approach to very special circumstances
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