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

A rapid, robust and widely applicable mutation detection scheme not requiring radioactivity or gel-based detection is introduced. It is a single-tube, competitive oligonucleotide single-base extension (COSBE) reaction using a pair of primers with the 3′-terminal base complementary to either the normal or mutant allele. Upon hybridization and addition of a polymerase and the nucleotide triphosphate corresponding to the next base after the primer, only those primers properly annealed (i.e., no 3′-terminal mismatch) are extended; products are resolved by molecular weight shifts as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (single-scan spectrum acquisition <<1 s). For the cystic fibrosis ΔF508 polymorphism, 28-mer “normal” (N) and 30-mer “mutant” (M) primers generate 29-mer (N+1) or 31-mer (M+1) products for homozygotes and both for heterozygotes. Since primer and product molecular weights are relatively low (<10 kDa) and the mass difference between these are a...