Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry.
While electrospray ionization mass spectrometry has shown great potential for the identification of genotypes in DNA sequences amplified by polymerase chain reaction (PCR), the quantitative determination of allele frequencies remains challenging because of the presence of cationic adducts in the mass spectra which severely impairs accuracy of quantitation. We report here on the elaboration of an optimized desalting protocol for ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) of PCR amplicons which facilitates the genotyping of single nucleotide polymorphisms (SNPs). Chromatographic purification at temperatures between 50 and 70 degrees C using monolithic reversed-phase columns and acetonitrile gradients in aqueous, 20-30 mmol/l butyldimethylammonium bicarbonate enabled the mass spectrometric analysis of nucleic acid solutions containing up to 1.7 mol/l sodium chloride. A further improvement in removal of metal cations was achieved upon the addition of 5-10 mmol/l ethylenediaminetetraacetic acid (EDTA) to the sample solution prior to liquid chromatography. ICEMS was used for the semi-quantitative genotyping of SNPs amplified from the tetraploid genome of potato cultivars. Using a quadrupole ion trap mass spectrometer, allele frequencies were determined with an accuracy of 2-9% by measuring the relative intensities of the signals corresponding to the molecular mass of each of the alleles in the deconvoluted mass spectra. ICEMS results correlated well with those obtained by pyrosequencing, single nucleotide primer extension, and conventional dideoxy sequencing.
Oberacher
Herbert
H
Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria.
Parson
Walther
W
Hölzl
Georg
G
Oefner
Peter J
PJ
Huber
Christian G
CG
D002851:Chromatography, High Pressure Liquid; D041322:Chromosomes, Human, Y; D018736:DNA Adducts / Q000737:chemistry*; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D018736:DNA Adducts