Abstract
Optimal temperature conditions for the detection of 28 known mutations on 15 exons of the human cystic fibrosis transmembrane conductance regulator gene by single strand conformation polymorphism analysis using the Diagen TGGE Apparatus were established. This procedure was applied to the detection of unknown mutations in 58 non-deltaF508 chromosomes. Three novel mutations,-471del3 (5′ flanking region), 3171insC (exon 17a) and 4700(T)8/9 (3′ non-translated region) of the CFTR gene were found. Mutation 3171insC occured in conjunction with the delta F508 mutation on the other allele of a child presenting with severe pathology. Mutation -471 del3 has so far only been found in one healthy individual and her father, and 4700(T)8/9 is a DNA sequence polymorphism.
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Cotton RGH, Rodriges NR, Campbell RD (1988) Reactivity of cytosine and thymine in single base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci USA 85:4397–4401
Coutelle C (1991) New DNA analysis techniques. Biomed Biochim Acta 50:1–3
Coutelle C, Brückner R, Grade K, Behrens F, Gedschold J, Hein J, Szibor R, Bauer I, Brock J, Graupner I, Urner U, Leucht B (1992) Prevalence of cystic fibrosis mutations in the East German population. Hum Mutat 1:109–112
Dean M, White MB, Amos J, Gerrard B, Steward C. Khaw K-T, Leppert M (1990) Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients. Cell 61:863–870
Grompe M (1993) The rapid detection of unknown mutations in nucleic acids. Nature Genet 5:111–116
Hayashi K (1992) PCR-SSCP: a method for detection of mutations. Genet Anal Tech Appl 9:73–79
Hongyo T, Buzard GS, Calvert RJ, Weghorst CM (1993) Cold SSCP: a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses. Nucleic Acids Res 21:3637–3642
Kerem B-S. Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui L-C (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245:1073–1080
Lerman SL, Silverstein K (1987) Computerized simulation of DNA melting and its application to denaturing gel electrophoresis. Methods Enzymol 155:482–501
Orita M, Suzuki Y, Sekiya T, Hayashi K (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874–879
Riordan JR, Rommens JM, Kerem B-S, Alon N. Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou J-L, Drumm ML, Iannuzzi MC, Collins FS, Tsui L-C (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066–1073
Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW, Stone EM (1993) The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions. Genomics 16:325–332
Tsui L-C (1992) Mutations and sequence variations detected in the cystic fibrosis transmembrane conductance regulator (CFTR) gene: a report of the cystic fibrosis analysis consortium. Hum Mutat 1:197–203
Walsh PS, Metzger DA, Higuchi R (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing or forensic material. Biotechniques 10:506–513
Zielenski J, Rozmahel R. Bozon D, Kerem B-S, Grzelczak Z, Riordan JR, Rommens J, Tsui L-C (1991) Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Genomics 10:214–228
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Grade, K., Grunewald, I., Graupner, I. et al. Identification of three novel mutations in the CFTR gene using temperature-optimized non-radioactive conditions for SSCP analysis. Hum Genet 94, 154–158 (1994). https://doi.org/10.1007/BF00202861
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DOI: https://doi.org/10.1007/BF00202861