Competitive Oligonucleotide Single-Base Extension Combined with Mass Spectrometric Detection for Mutation Screening

摘要: A rapid, robust and widely applicable mutation detection scheme not requiring radioactivity or gel-based is introduced. It a single-tube, competitive oligonucleotide single-base extension (COSBE) reaction using pair of primers with the 3'-terminal base complementary to either normal mutant allele. Upon hybridization addition polymerase nucleotide triphosphate corresponding next after primer, only those properly annealed (i.e., no mismatch) are extended; products resolved by molecular weight shifts as determined matrix-assisted laser desorption ionization time-of-flight mass spectrometry (single-scan spectrum acquisition < 1 s). For cystic fibrosis delta F508 polymorphism, 28-mer "normal" (N) 30-mer "mutant" (M) generate 29-mer (N + I) 31-mer (M 1) for homozygotes both heterozygotes. Since primer product weights relatively low (< 10 kDa) difference between these at least that single approximately 300-Da unit, low-resolution spectrometer suitable such measurements.