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 delta F508 polymorphism, 28-mer "normal" (N) and 30-mer "mutant" (M) primers generate 29-mer (N + I) 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 at least that of a single approximately 300-Da nucleotide unit, a low-resolution mass spectrometer is suitable for such measurements.
Higgins
G S
GS
SEQUENOM Instruments GmbH, Hamburg, Germany.
Little
D P
DP
Köster
H
H
D000483:Alleles; D001482:Base Composition; D001667:Binding, Competitive; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D006801:Humans; D013058:Mass Spectrometry; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
C505032:CFTR protein, human; D017931:DNA Primers; D009841:Oligonucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator