Single-nucleotide polymorphisms: analysis by mass spectrometry

Nat Protoc. 2006;1(4):1761-71. doi: 10.1038/nprot.2006.257.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylation
  • DNA Primers / chemistry
  • Exonucleases
  • Genotype
  • Microarray Analysis
  • Oligonucleotides / isolation & purification
  • Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide*
  • Solid Phase Extraction
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods*
  • Transferases

Substances

  • DNA Primers
  • Oligonucleotides
  • Transferases
  • Exonucleases
  • spleen exonuclease