ABCMdb

PMID: 7477013

Rossetti S, Corra S, Biasi MO, Turco AE, Pignatti PF
Comparison of heteroduplex and single-strand conformation analyses, followed by ethidium fluorescence visualization, for the detection of mutations in four human genes.
Mol Cell Probes. 1995 Jun;9(3):195-200., [PubMed]

Sentences

No. Mutations Sentence Comment

22 ABCC7 p.Arg75Gln 18 The second upper primer developed for mutation R75Q detection (UP-40) was the following: GAA- TGGGATAGAGAGCTGGC'I-rC, corresponding to nucleotides 298 to 320 of the CFTR gene.~9Flanking primers for each target sequence were synthesized with an Applied Biosystem 391 DNA synthesizer PCR-MATE EP,and PCRwas performed with a Perkin Elmer thermal cycler, following published data.i° Genomic DNA was used for exon amplification of CFTR and COL4A5 genes. Login to comment 40 ABCC7 p.Arg334Trp 7 and 8: normal control and R334W (CFTR)carrier, respectively. Login to comment 41 ABCC7 p.Arg347Pro 9 and 10: normal control and R347P (CFTR)carrier, respectively. Molecular weight marker: 1 kb ladder (Gibco BRL).Arrowhead: double stranded (ds) DNA (230 bp), 7-10. Login to comment 45 ABCC7 p.Arg75Gln Heteroduplex bands are visible in all samples, except the G177R and the R75Q mutations in lanes 3 and 5, respectively, where only a large homoduplex band is present. Login to comment 52 ABCC7 p.Arg75Gln The only mutation not detected with either method was R75Q located in the CFTR gene. Login to comment 57 ABCC7 p.Arg347Pro ABCC7 p.Arg75Gln 1: 1271delC (COL4A5), male, 2: R344W (CFTR), carrier, 3:G177R (COL4A5), male, 4: 2940/ 2943delA (COL4A5), male, 5: R75Q (CFTR),carrier, 6: R347P (CFTR),carrier. Login to comment 61 ABCC7 p.Arg334Trp ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Arg347Pro ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln Mutations analysed by SSCAEand HAE: reproducibility scores Gene Mutation Exon PCR Sequence SSCAE SSCAE HAE (reference) size modification +4°C +20°C (A) Amplified fragment size range: 68-230 bp CFTR R334W 7 230 C~T + + + + + + (1) CFTR R347P 7 230 G--*C + + + + + + (2) CFTR R75Q 3 176 G~A - - - (UP-1) (3) CFTR R75Q 3 213 G~A + + + (UP-40) CFTR 2183AAJG 13 145 AA~C + + + - + + (4) COL4A5 2940/2943 34 146 Delta A + + + + + delA (5) COL4A5 1272delC 42 181 Delta C + + + + + (6) COL4A5 G325E 17 68 G~A + + + (7) COL4A5 G17R* 9 121 G~C + + + + - COL4A5 G177R* 9 121 G-,C + + + + - COL4A5 G54D* 3 230 G~A + + + - + + (B) Amplified fragment size range: 257-426 bp HEX B P405L 11 426 C~T - - (8) HEX B 929delT* 8 426 Delta T - - HEX B C317Y* 8 270 G~A - - COL1 A2 G586V 33-37 257 G~T - + (9) COL1A2 G640C 33-37 257 G--.T - - (10) + + ++ + + + + =very good (515repeats); + =good (4•5 repeats); +- =sufficient (315repeats); - =not sufficient(0, or 2/5 repeats). Login to comment 108 ABCC7 p.Arg75Gln FluorogramofSSCAEand HAE analysis of mutation R75Q (CFTR)with the use of different PCR primers. Login to comment 111 ABCC7 p.Arg75Gln FluorogramofSSCAEand HAE analysis of mutation R75Q (CFTR)with the use of different PCR primers. Login to comment