Multiplex genotyping of the human beta2-adrenergic receptor gene using solid-phase capturable dideoxynucleotides and mass spectrometry.
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.
Kim
Sobin
S
Laboratory of DNA Sequencing and Chemical Biology, Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Shi
Shundi
S
Bonome
Tomás
T
Ulz
Michael E
ME
Edwards
John R
JR
Fodstad
Heidi
H
Russo
James J
JJ
Ju
Jingyue
J
D001483:Base Sequence; D019921:Biotinylation; D017931:DNA Primers; D015224:Dideoxynucleosides / Q000032:analysis* / Q000737:chemistry; D005838:Genotype; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D017931:DNA Primers; D015224:Dideoxynucleosides; D018343:Receptors, Adrenergic, beta-2