Needle-in-a-haystack detection and identification of base substitution mutations in human tissues.
Background and induced germline mutagenesis and other genotoxicity studies have been hampered by the lack of a sufficiently sensitive technique for detecting mutations in a small cluster of cells or a single cell in a tissue sample composed of millions of cells. The most frequent type of genetic alteration is intragenic. The vast majority of oncogenic mutations in human and mammalian cancer involves only single base substitutions. We have developed universally applicable techniques that not only provide the necessary sensitivity and specificity for site specific mutagenesis studies, but also identify the point mutation. The exponential amplification procedures of polymerase chain reaction (PCR) and ligase chain reaction (LCR) have been combined with restriction endonuclease (RE) digestion to enable the selective enrichment and detection of single base substitution mutations in human oncogenic loci at a sensitivity of one mutant in more than 10(7) wild type alleles. These PCR/RE/LCR procedures have been successfully designed and used for codons 12 and 248 of the Ha-ras and p53 genes, respectively, both of which contain a natural MspI restriction endonuclease recognition sequence. These procedures have also been adapted for the detection and identification of mutations in oncogenic loci that do not contain a natural restriction endonuclease recognition sequence. Using PCR techniques, a HphI site was incorporated into the codons 12/13 region of the human N-ras gene, which was then used for the selective enrichment of mutants at this oncogenic locus. These PCR/RE/LCR procedures for base substitution mutations in codon 12 of the N-ras gene were found to have the sensitivity of detection of at least one mutant allele in the presence of the DNA equivalent of 10(6) wild type cells. Only one peripheral blood leukocyte DNA specimen out of nine normal individuals displayed an observable Ha-ras mutation that was present at frequency between 10(-5) and 10(-6). These PCR/RE/LCR techniques for detecting and identifying base substitution mutations are universally applicable to almost any locus or base site within the human or animal genome. With the added advantage of the adjustability of both the amount of DNA (number of genomes) to be tested and the sensitivity (10(-2) to 10(-7)) of the assay selection or enrichment procedures, these PCR/RE/LCR techniques will be useful in addressing a broad range of important questions in mutagenesis and carcinogenesis.
Wilson
V L
VL
Institute of Environmental Studies, Louisiana State University, Baton Rouge, LA 70803, USA. monster77@aol.com
Wei
Q
Q
Wade
K R
KR
Chisa
M
M
Bailey
D
D
Kanstrup
C M
CM
Yin
X
X
Jackson
C M
CM
Thompson
B
B
Lee
W R
WR
D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D016158:Genes, p53 / Q000235:genetics; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004273:DNA, Neoplasm; D019003:Deoxyribonuclease HpaII; D011088:DNA Ligases