High level multiplex genotyping by MALDI-TOF mass spectrometry.
A primer extension assay is used to perform highly multiplexed genotyping of single nucleotide polymorphisms (SNPs) present in genomic DNA amplified by a multiplex PCR. The assay uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to accurately measure the masses of short oligonucleotide primers extended by a single dideoxynucleotide. The multiplexed genotyping assays rely on the natural molecular weight differences of DNA bases. By careful analysis of primer composition complementary to the target, or by judicious addition of one or more noncomplementary 5' bases to the genotyping primers, mass spectra of interleaved genotyping products can be generated with no ambiguity in allele assignment. Using a model multiplex PCR system, we demonstrate the ability to perform 12-fold multiplex SNP analysis.
Ross
P
P
PerSeptive Biosystems, Framingham, MA 01701, USA. Philip_Ross@pbio.com
Hall
L
L
Smirnov
I
I
Haff
L
L
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D005838:Genotype; D006801:Humans; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D012016:Reference Values; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D017931:DNA Primers; D004247:DNA