title: Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry.
abstract: A new method for the reliable identification of localized variations in DNA by detection of associated diagnostic products with matrix-assisted laser desorption ionization time-of-flight mass spectrometry is described. The diagnostic products are generated by the primer oligo base extension (PROBE) reaction, which requires a single detection primer complementary to a region down-stream of a target strand's variable site. On addition of a polymerase, three dNTPs, and the fourth nucleotide in dideoxy form, the primer is extended through the mutation region until the first ddNTP is incorporated; the mass of the extension products determines the composition of the variable site. Tests for five cystic fibrosis mutations, including two exon 11 sites measured in a biplex reaction, and for differentiating between three common alleles of the poly(T) tract at the intron 8 splice acceptor site of the CFTR gene are presented. All experimental steps required for PROBE are amenable to the high degree of automation desirable for a high-through-put diagnostic setting. Furthermore, it requires no fluorescent, chemiluminescent, or radioactive labeling; the mass signals measured offer a far more analytically definitive signal, leading in all cases to high-quality unambiguous and easily interpreted results.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Hamburg, Germany. abraun01@aol.com
authors.forename: A
authors.initials: A
authors.lastname: Braun
authors.forename: D P
authors.initials: DP
authors.lastname: Little
authors.forename: H
authors.initials: H
authors.lastname: Köster
mesh_terms: D000483:Alleles; D001483:Base Sequence; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D017931:DNA Primers; D003854:Deoxyribonucleotides; D015224:Dideoxynucleosides; D005091:Exons; D006801:Humans; D007438:Introns; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation; D012326:RNA Splicing
chemical_list: C505032:CFTR protein, human; D017931:DNA Primers; D003854:Deoxyribonucleotides; D015224:Dideoxynucleosides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator