Analysis of single nucleotide polymorphisms by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

A method for typing single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described, in which a mass-tagged dideoxynucleoside triphosphate is employed in a primer extension reaction in place of an unmodified dideoxynucleoside triphosphate (ddNTP). The increased mass difference due to the presence of the mass-tag greatly facilitates the accurate identification of the added nucleotide, and is particularly useful for typing heterozygous samples. Twenty commercially available mass-tagged dideoxynucleoside triphosphates were screened for amenability to incorporation by AmpliTaq FS and ThermoSequenase DNA polymerases in single nucleotide primer extension (SNuPE) reactions. Several sample preparation and purification methods were also examined and compared. Float dialysis was found to be a simple, versatile, and effective method for purification of the extension products. High specificity and sensitivity were obtained, and all six possible biallelic SNP heterozygotes were determined accurately using a 44-mer synthetic oligonucleotide target DNA as a model system. Further validation of the method was demonstrated in the analysis of five single-base mutations in exon IV of the human tyrosinase gene. Single nucleotide variations within 182-bp PCR amplicons amplified from three plasmid and three human genomic DNA samples were genotyped at five variable positions, with results in 100% concordance with conventional sequencing. Genotypes were determined accurately at five sequence-tagged sites (STSs).

Influence of Leukotriene Pathway Polymorphisms on Response to Montelukast in Asthma

“…Single nucleotide polymorphisms (SNPs) were genotyped via a Sequenom matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass array spectrometer (Sequenom, San Diego, CA), using a semiautomated primer design program (Spectro Designer; Sequenom) (30)(31)(32). In addition, the LTC4S C-444A promoter SNP was genotyped and the number of Sp1 binding motifs (5ЈGGGCGG3Ј) in the ALOX5 promoter was determined as previously described (16,33).…”

Automation for Genomics, Part Two: Sequencers, Microarrays, and Future Trends

“…In March 1998, SEQUENOM (San Diego, CA) (http://www.sequenom.com) sequenced 670 bases of the p53 gene using MALDI-TOF (Fu et al 1998). For review articles on mass spectrometry, see Clark et al (1999), Griffin et al (1999), Aebersold et al (2000), Deforce and Van den Eeckhout (2000), Fei and Smith (2000), Gatlin et al (2000), , Griffin and Smith (2000), Griffiths (2000), Guilhaus et al (2000), Jackson et al (2000), Johnston (2000), Li et al (2000), Roepstorff (2000), and Yates (2000). A new mass spectrometric technique, charge reduction electrospray mass spectrometry (CREMS), is described in Scalf et al (2000).…”

Rapid characterization of DNA oligomers and genotyping of single nucleotide polymorphism using nucleotide-specific mass tags

“…Whereas several MALDI-TOF MS-based methods have been introduced for genotyping SNPs, the primary strategy has been to generate SNP-containing products through primer extension for MS analysis (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Haff and Smirnov invented a PinPoint assay based on the annealing of a primer to the 5′-end upstream of the target polymorphic site (14,15).…”

“…This mass overlap also affects the efficiency and accuracy of the analysis of a number of polymorphic sites using multiple primers in any primer extension-based assay. Additional masses added to the ddNTPs can overcome the original mass stringency by shifting the mass of the extended primer (20,21). However, this still demands high instrumental resolution to resolve small mass changes involved in base substitutions, e.g., the 9 Da mass difference between an A and a T.…”