Single-nucleotide polymorphisms: analysis by mass spectrometry.
Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry has evolved as a powerful method for analyzing nucleic acids. Here we provide protocols for genotyping single-nucleotide polymorphisms (SNPs) by MALDI based on PCR and primer extension to generate allele-specific products. Furthermore, we present three different approaches for sample preparation of primer-extension products before MALDI analysis and discuss their potential areas of application. The first approach, the 'GOOD' assay, is a purification-free procedure that uses DNA-modification chemistry, including alkylation of phosphorothioate linkages in the extension primers. The other two approaches use either solid-phase extraction or microarray purification for the purification of primer-extension products. Depending on the reaction steps of the various approaches, the protocols take about 6-8 hours.
Sauer
Sascha
S
Max Planck Institute for Molecular Genetics, Department of Vertebrate Genomics, Ihnestrasse 63-73, 14195 Berlin-Dahlem, Germany. sauer@molgen.mpg.de
Reinhardt
Richard
R
Lehrach
Hans
H
Gut
Ivo G
IG
D000478:Alkylation; D017931:DNA Primers / Q000737:chemistry; D005092:Exonucleases; D005838:Genotype; D046228:Microarray Analysis; D009841:Oligonucleotides / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D052616:Solid Phase Extraction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014166:Transferases
D017931:DNA Primers; D009841:Oligonucleotides; D014166:Transferases; D005092:Exonucleases; C022650:spleen exonuclease