Multiplex genotyping of the human β2-adrenergic receptor gene using solid-phase capturable dideoxynucleotides and mass spectrometry

Sobin Kim, Shundi Shi, Tomás Bonome, Michael E. Ulz, John R. Edwards, Heidi Fodstad, James J. Russo, Jingyue Ju

Research output: Contribution to journalArticle

17 Scopus citations

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 β2-adrenergic receptor (β2AR) 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 β2AR 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.

Original languageEnglish
Pages (from-to)251-258
Number of pages8
JournalAnalytical Biochemistry
Volume316
Issue number2
DOIs
StatePublished - May 15 2003
Externally publishedYes

Keywords

  • Biotinylated dideoxynucleotides
  • MALDI-TOF mass spectrometry
  • Multiplex genotyping
  • SNP
  • Single base extension
  • β2-Adrenergic receptor gene

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