Analysis of sequence variations in several human genes using phosphoramidite bond DNA fragmentation and chip-based MALDI-TOF.
The challenge in the postgenome era is to measure sequence variations over large genomic regions in numerous patient samples. This massive amount of work can only be completed if more accurate, cost-effective, and high-throughput solutions become available. Here we describe a novel DNA fragmentation approach for single nucleotide polymorphism (SNP) discovery and sequence validation. The base-specific cleavage is achieved by creating primer extension products, in which acid-labile phosphoramidite (P-N) bonds replace the 5' phosphodiester bonds of newly incorporated pyrimidine nucleotides. Sequence variations are detected by hydrolysis of this acid-labile bond and MALDI-TOF analysis of the resulting fragments. In this study, we developed a robust protocol for P-N-bond fragmentation and investigated additional ways to improve its sensitivity and reproducibility. We also present the analysis of several human genomic targets ranging from 100-450 bp in length. By using a semiautomated sample processing protocol, we investigated an array of SNPs within a 240-bp segment of the NFKBIA gene in 48 human DNA samples. We identified and measured frequencies for the two common SNPs in the 3'UTR of NFKBIA (separated by 123 bp) and then confirmed these values in an independent genotyping experiment. The calculated allele frequencies in white and African American groups differed significantly, yet both fit Hardy-Weinberg expectations. This demonstrates the utility and effectiveness of PN-bond DNA fragmentation and subsequent MALDI-TOF MS analysis for the high-throughput discovery and measurement of sequence variations in fragments up to 0.5 kb in length in multiple human blood DNA samples.
Smylie
Kevin J
KJ
Sequenom Inc., San Diego, California, 92121, USA.
Cantor
Charles R
CR
Denissenko
Mikhail F
MF
D020413:3' Untranslated Regions / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D053480:Cholesterol Ester Transfer Proteins; D004247:DNA / Q000032:analysis*; D053938:DNA Fragmentation / Q000235:genetics*; D005796:Genes / Q000235:genetics*; D014644:Genetic Variation / Q000235:genetics*; D005838:Genotype; D006023:Glycoproteins; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics; D007473:Ion Channels; D026901:Membrane Transport Proteins / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D000071246:Uncoupling Protein 2
D020413:3' Untranslated Regions; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D006023:Glycoproteins; D020672:I-kappa B Proteins; D007473:Ion Channels; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; C000606204:NFKBIA protein, human; D009943:Organophosphorus Compounds; D000071246:Uncoupling Protein 2; C434331:phosphoramidite; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA