Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry

摘要: A new method for the reliable identification of localized variations in DNA by detection associated diagnostic products with matrix-assisted laser desorption ionization time-of-flight mass spectrometry is described. The are generated primer oligo base extension (PROBE) reaction, which requires a single complementary to region down-stream target strand's variable site. On addition polymerase, three dNTPs, and fourth nucleotide dideoxy form, extended through mutation until first ddNTP incorporated; determines composition Tests five cystic fibrosis mutations, including two exon 11 sites measured biplex differentiating between common alleles poly(T) tract at intron 8 splice acceptor site CFTR gene presented. All experimental steps required PROBE amenable high degree automation desirable high-through-put setting. Furthermore, it no fluorescent, chemiluminescent, or radioactive labeling; signals offer far more analytically definitive signal, leading all cases high-quality unambiguous easily interpreted results.