Genotypic Analysis of Mutations in Taq I Restriction Recognition Sites by Restriction Fragment Length Polymorphism/Polymerase Chain Reaction

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 3; pp. 890 - 894
• Main Authors: Sandy, Martha S., Chiocca, Susanna M., Cerutti, Peter A.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01-02-1992; National Acad Sciences; National Academy of Sciences
• Subjects: 550201 - Biochemistry- Tracer Techniques; ANIMALS; Base Sequence; BASIC BIOLOGICAL SCIENCES; Biochemistry; Biological and medical sciences; Cellular biology; Deoxyribonucleases, Type II Site-Specific - metabolism; Diverse techniques; DNA; DNA BASE TRANSITIONS; DNA POLYMERASES; DNA SEQUENCING; DNA-ASE; ENDONUCLEASES; ENZYMES; EPIDEMIOLOGY; ESTERASES; Exons; Fundamental and applied biological sciences. Psychology; GENE AMPLIFICATION; GENE MUTATIONS; GENES; Genes, ras; Genetic mutation; Genetics; GENOTYPE; HYDROLASES; MAMMALS; MAN; Molecular and cellular biology; Molecular Sequence Data; Mutation; MUTATIONS; NUCLEIC ACIDS; Nucleotides; NUCLEOTIDYLTRANSFERASES; Oligodeoxyribonucleotides - chemistry; Oligonucleotide probes; OLIGONUCLEOTIDES; ONCOGENES; ORGANIC COMPOUNDS; PHOSPHODIESTERASES; PHOSPHORUS-GROUP TRANSFERASES; point mutation; Polymerase chain reaction; Polymerase Chain Reaction - methods; POLYMERASES; Polymorphism, Genetic; PRIMATES; Product standards; PROTEINS; restriction fragment length polymorphism; RFLPS; Sequencing; STRUCTURAL CHEMICAL ANALYSIS; TRANSFERASES; VERTEBRATES; Human; Polymerase chain reaction; Site specificity; Restriction fragment length polymorphism; Enzyme; C-Onc gene; Endodeoxyribonuclease TaqI; Mutation; Method; Protooncogene; Recognition; Quantitative analysis
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.3.890

Point mutations in somatic cells play a role in the etiology of several classes of human pathologies. Experimental procedures are required that allow the detection and quantitation of such mutations in disease-related genes in tissue biopsy samples without the need for the selection of mutated cells. We describe the genotypic analysis of single base pair mutations in the Taq I endonuclease recognition sequence TCGA, residues 2508-2511 of exon 2 of the human c-H-ras1 gene, by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) approach. The high thermostability of Taq I endonuclease allows the continuous removal of eventual residual wild-type sequences during the thermocycling of the PCR and reduces polymerase errors in the final RFLP/PCR product to a minimum. As few as five copies of a mutant standard containing two base pair changes in the chosen Taq I site could be rescued from 108copies of wild-type DNA. Taq I RFLP/PCR holds promise for the monitoring of mutations in biochemical epidemiology.