Multiplex genotyping of single nucleotide polymorphisms using MALDI-TOF mass spectrometry

Abstract

We report a multiplex analysis approach using mass-tagged extension primers in single base extension for genotyping of single nucleotide polymorphism (SNP). In this method, oligonucleotide primers that are specific to polymorphic sites in the DNA template are simultaneously extended with each biotin-ddNTP by DNA polymerase to generate 3’-biotinylated DNA products in each four tube. These products in each tube are captured by streptoavidin-coated magnetic beads while unextended primers and other components are washed away. These captured extended primers which have one deoxyuridine sequence between 5’-mass-tag region and 3’-template recognition region are cut by uracil-DNA glycosylase to generate liberal oligonucleotides that have distinct mass according to specific SNP sites. The liberal oligonucleotides which code specific SNP sites in each tube are identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We successfully analyzed 50 SNP loci at the same time.