Multiplex genotyping of the human beta2-adrenergic receptor gene using solid-phase capturable dideoxynucleotides and mass spectrometry |
| |
Authors: | Kim Sobin Shi Shundi Bonome Tomás Ulz Michael E Edwards John R Fodstad Heidi Russo James J Ju Jingyue |
| |
Institution: | Laboratory of DNA Sequencing and Chemical Biology, Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA. |
| |
Abstract: | Previously, we established the feasibility of using solid phase capturable (SPC) dideoxynucleotides to generate single base extension (SBE) products which were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for multiplex genotyping, an approach that we refer to as SPC-SBE. We report here the expanding of the SPC-SBE method as a single-tube assay to simultaneously detect 20 single nucleotide variations in a model system and 3 single nucleotide polymorphisms (SNPs) in the human beta2-adrenergic receptor (beta2AR) gene. Twenty primers were designed to have a sufficient mass difference between all extension products for accurate detection of nucleotide variants of the synthetic templates related to the p53 gene. These primers were extended simultaneously in a single tube with biotin-ddNTPs to generate 3(')-biotinylated DNA products, which were first captured by streptavidin-coated magnetic beads and then released from the beads and analyzed with MALDI-TOF MS. This approach generates a mass spectrum free of primer peaks and their associated dimers, increasing the scope of multiplexing SNPs. We also simultaneously genotyped 3 SNPs in the beta2AR gene (5(')LC-Cys19Arg, Gly16Arg, and Gln27Glu) from the genomic DNA of 20 individuals. Comparison of this approach with direct sequencing and the restriction fragment length polymorphism method indicated that the SPC-SBE method is superior for detecting nucleotide variations at known SNP sites. |
| |
Keywords: | SNP MALDI-TOF mass spectrometry Biotinylated dideoxynucleotides Single base extension Multiplex genotyping β2-Adrenergic receptor gene |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|