MUTATIONAL ANALYSIS BY A COMBINED APPLICATION OF THE MULTIPLE RESTRICTION FRAGMENT-SINGLE STRAND CONFORMATION POLYMORPHISM AND THE DIRECT LINEAR AMPLIFICATION DNA SEQUENCING PROTOCOLS

被引:33
作者
LEE, HH [1 ]
LO, WJ [1 ]
CHOO, KB [1 ]
机构
[1] VET GEN HOSP, DEPT MED RES, RECOMBINANT DNA LAB, TAIPEI 11217, TAIWAN
关键词
D O I
10.1016/0003-2697(92)90437-C
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We describe here an improved procedure for polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) for rapid mutational detection. To circumvent the restriction of having to analyze relatively short PCR fragments, restriction endonucleases were used to cleave a longer PCR product and the mixture of fragments was analyzed directly in SSCP gel electrophoresis. This multiple restriction fragment (MRF)-SSCP protocol was demonstrated by the detection of a 4-bp deletion in codons 41-42 and a point mutation in the IVS-2 sequence of the human β-globin gene. The MRF-SSCP or the standard SSCP protocol was then combined with the linear amplification DNA sequencing (LADS) procedure for direct analysis of the PCR products without further purification for an exact characterization of the mutations detected. In the LADS analysis, homo- or heterozygosity of a mutation was easily distinguished by the appearance of a single-or double-lane band in the sequencing gel. The choice of isotope used and different labeling methods were compared and were found, in some cases, to produce SSCP patterns of different complexities. The combined MRFSSCP/LADS protocol permits rapid mutational analysis of a large number of clinical samples using only very small amounts of materials and can easily be adopted for nonisotopic clinical applications. © 1992.
引用
收藏
页码:289 / 293
页数:5
相关论文
共 23 条
[1]   MOLECULAR CHARACTERIZATION OF BETA-GLOBIN GENE-MUTATIONS IN PATIENTS WITH BETA-THALASSEMIA INTERMEDIA IN SOUTH CHINA [J].
ANTONARAKIS, SE ;
KANG, J ;
LAM, VMS ;
TAM, JWO ;
LI, AMC .
BRITISH JOURNAL OF HAEMATOLOGY, 1988, 70 (03) :357-361
[2]   FAST AND SENSITIVE SILVER STAINING OF DNA IN POLYACRYLAMIDE GELS [J].
BASSAM, BJ ;
CAETANOANOLLES, G ;
GRESSHOFF, PM .
ANALYTICAL BIOCHEMISTRY, 1991, 196 (01) :80-83
[3]   DETECTION OF SICKLE-CELL BETA-S-GLOBIN ALLELE BY HYBRIDIZATION WITH SYNTHETIC OLIGONUCLEOTIDES [J].
CONNER, BJ ;
REYES, AA ;
MORIN, C ;
ITAKURA, K ;
TEPLITZ, RL ;
WALLACE, RB .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1983, 80 (01) :278-282
[4]   REACTIVITY OF CYTOSINE AND THYMINE IN SINGLE-BASE-PAIR MISMATCHES WITH HYDROXYLAMINE AND OSMIUM-TETROXIDE AND ITS APPLICATION TO THE STUDY OF MUTATIONS [J].
COTTON, RGH ;
RODRIGUES, NR ;
CAMPBELL, RD .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (12) :4397-4401
[5]   DIRECT SEQUENCING OF ENZYMATICALLY AMPLIFIED HUMAN GENOMIC DNA [J].
ENGELKE, DR ;
HOENER, PA ;
COLLINS, FS .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (02) :544-548
[6]   DNA FRAGMENTS DIFFERING BY SINGLE BASE-PAIR SUBSTITUTIONS ARE SEPARATED IN DENATURING GRADIENT GELS - CORRESPONDENCE WITH MELTING THEORY [J].
FISCHER, SG ;
LERMAN, LS .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1983, 80 (06) :1579-1583
[7]   PURIFICATION OF DNA FROM FORMALDEHYDE FIXED AND PARAFFIN EMBEDDED HUMAN-TISSUE [J].
GOELZ, SE ;
HAMILTON, SR ;
VOGELSTEIN, B .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1985, 130 (01) :118-126
[8]   GENERATION OF SINGLE-STRANDED-DNA BY THE POLYMERASE CHAIN-REACTION AND ITS APPLICATION TO DIRECT SEQUENCING OF THE HLA-DQA LOCUS [J].
GYLLENSTEN, UB ;
ERLICH, HA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (20) :7652-7656
[9]   USE OF LABELED PRIMERS IN POLYMERASE CHAIN-REACTION (LP-PCR) FOR A RAPID DETECTION OF THE PRODUCT [J].
HAYASHI, K ;
ORITA, M ;
SUZUKI, Y ;
SEKIYA, T .
NUCLEIC ACIDS RESEARCH, 1989, 17 (09) :3605-3605
[10]   TISSUE EXTRACTION OF DNA AND RNA AND ANALYSIS BY THE POLYMERASE CHAIN-REACTION [J].
JACKSON, DP ;
LEWIS, FA ;
TAYLOR, GR ;
BOYLSTON, AW ;
QUIRKE, P .
JOURNAL OF CLINICAL PATHOLOGY, 1990, 43 (06) :499-504