Detection of p53 gene mutations: analysis by single-strand conformation polymorphism and Cleavase fragment length polymorphism.
We have generated a collection of clones containing single point mutations within the exon 5-9 hot spot regions of the p53 gene by using polymerase chain reaction (PCR) to amplify select regions of the gene from characterized cell lines. These clones were then used to address the sensitivity of mutation detection using slab-gel single-strand conformation polymorphism (SSCP) and Cleavase fragment length polymorphism (CFLP) assay systems. Both methods exhibited high sensitivities for the detection of mutations in cloned p53 mutations in this study: 97% for CFLP and 94% for SSCP. In addition to resulting in higher sensitivity of mutation detection, CFLP has the capability to analyze longer fragments. In this study, CFLP identified five intronic mutations which were not investigated in the exon-specific SSCP assay. These results agree with those found elsewhere and demonstrate that CFLP scanning can have practical advantages when used for the identification of sequence alterations within the p53 gene.
O'Connell
C D
CD
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. coc@nist.gov
Atha
D H
DH
Oldenburg
M C
MC
Tian
J
J
Siebert
M
M
Handrow
R
R
Grooms
K
K
Heisler
L
L
de Arruda
M
M
D016158:Genes, p53; D006801:Humans; D009154:Mutation; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods