Highly sensitive mutation screening by REF with low concentrations of urea: A blinded analysis of a 2-kb region of the p53 gene reveals two common haplotypes.
Restriction endonuclease fingerprinting (REF), a hybrid modification of single-strand conformation polymorphism (SSCP) and restriction endonuclease digestion, has been used previously to detect mutations in 1- to 2-kb segments of DNA. This paper demonstrates that fragment resolution, and thus sensitivity of REF, can be markedly improved by electrophoresis under partially denaturing, rather than nondenaturing, conditions, for genes with a high G+C content. A 2. 1-kb segment of the p53 tumor suppressor gene (54.5% G+C) containing exons 5-9, including the intervening introns, was screened in a blinded analysis of 48 samples from human breast tumors containing known wild-type or mutant p53 genes. In gels containing 0.5 M urea, 97% of the mutant samples were detected correctly, and more than 80% of the mutations were localized within a 200-bp region. In the process of this methodological analysis, it was discovered that: (1) there are two common and four uncommon haplotypes; (2) the two common haplotypes occurred in the three races examined, suggesting an ancient origin; and (3) haplotype II is of substantially higher frequency in the Chinese relative to Japanese (P = 0.023) and Caucasians (P = 0.005). Two other improvements in the REF procedure included (1) the selection of an optimal set of restriction endonucleases by new software (REF Select) developed recently in our laboratory; and (2) the addition of an oligonucleotide "tail," containing two recognition sequences for restriction endonucleases, to the PCR primers to prevent coterminal fragments at the end of amplified products. These modifications facilitate the use of REF for efficient and sensitive mutation screening in p53 and other genes with a high G+C content.
Feng
J
J
Department of Molecular Genetics, City of Hope National Medical Center and Beckman Research Institute, Duarte, California 91010, USA.
Buzin
C H
CH
Tang
S H
SH
Scaringe
W A
WA
Sommer
S S
SS
D001943:Breast Neoplasms / Q000208:ethnology / Q000235:genetics*; D016172:DNA Fingerprinting / Q000379:methods*; D016158:Genes, p53 / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D007438:Introns; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014481:United States; D014508:Urea
D014508:Urea