Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry

Abstract

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°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

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

“…A PS-DVB capillary monolith column 21,[24][25][26][27][28][29][30][31][32][33] , a C 18 reverse-phase particulate silica column 23,40,41 and a hydrophobic interaction chromatography (HILIC) column 42 have been used for the LC/ESI-MS analysis of oligonucleotides. Among them, the capillary monolith column is superior to the others in terms of separation capacity, however, the capillary monolith column used in past studies was made in-house and operated at a low flow rate (2 µl/min), which requires instrumentation dedicated to micro LC.…”

“…LC provides an on-line separation of the analytes from coexisting substances and does not require a laborious sample preparation prior to ionization 21,22 . Application of LC/ESI-MS genotyping for differentiation of pathogen species, single-nucleotide polymorphisms and short tandem repeats has been reported using a capillary polymer-monolith column that can separate longer oligonucleotides 1,21,[24][25][26][27][28][29][30][31][32][33] .…”

Phosphorothioate Oligonucleotide Quantification by μ-Liquid Chromatography-Mass Spectrometry

“…Chromatographic separations were performed at 70°C because elevated temperatures give rise to improved separation efficiency and have a positive effect on desalting efficiency (41). Phosphorothioate oligonucleotides can undergo partial desulfurization getting in contact with oxidizing agents (42)(43)(44).…”

Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS

“…Even moderate concentrations of salts are known to detract from the ionization efficiency of nucleic acids and need to be removed (for a detailed discussion of this problem, please refer to references [1,8,9]. In this context, chromatography was shown to be one of the most efficient nucleic acid purification techniques available [9].…”

“…In this context, chromatography was shown to be one of the most efficient nucleic acid purification techniques available [9]. Due to the online hyphenation of chromatography to ESI-MS, the steps necessary to prepare genomic DNA samples for mass spectrometric analysis could be limited to PCR.…”