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


CONCLUSIONS
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.
Research & Development Center, GeneMatrix, Inc., Yongin, 446-913, South Korea. sunphong@genematrix.net
Sun Pyo
SP
Hong
Seung Il
SI
Ji
Hwanseok
H
Rhee
Soo Kyeong
SK
Shin
Sun Young
SY
Hwang
Seung Hwan
SH
Lee
Soong Deok
SD
Lee
Heung-Bum
HB
Oh
Wangdon
W
Yoo
Soo-Ok
SO
Kim
D000483:Alleles; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific; D005787:Gene Frequency; D005821:Genetic Techniques; D006239:Haplotypes; D006801:Humans; D007723:Korea; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D017931:DNA Primers; D004247:DNA; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D015252:Deoxyribonucleases, Type II Site-Specific