Non-radioactive detection of K-ras mutations by nested allele specific PCR and oligonucleotide hybridization.
The development of methods to detect point mutations has been rapid over the recent years. In human colon tumours, a significant percentage of mutations are found in the K-ras gene. Faster and more sensitive methods for detection of these mutations are important for patient management and treatment. The author has extended the specificity of allele amplification of point mutations by using mismatch oligonucleotide primers in a polymerase chain reaction (PCR). It is shown that in colon tumours, the sensitivity of detecting a mutation is significantly higher when K-ras exon 1 is amplified prior to PCR with allele specific primers (mismatch PCR). It is shown that allele specific oligonucleotide probes which are non-radioactively labelled could be used to detect these point mutations. By utilizing this two-step PCR, K-ras codon 12 mutations were studied in 10 colon carcinoma cell lines and 25 colon tumours. By combining the two-step PCR together with non-radioactively labelled oligonucleotide probes, the detection of point mutations is both accurate and rapid.
Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, DC 20307, USA.
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Paranavitana
D000409:Alanine; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid; D001943:Breast Neoplasms / Q000235:genetics; D003062:Codon; D003110:Colonic Neoplasms / Q000235:genetics; D004252:DNA Mutational Analysis; D017931:DNA Primers; D005260:Female; D011905:Genes, ras; D005998:Glycine; D006579:Heterozygote; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D003062:Codon; D017931:DNA Primers; D015345:Oligonucleotide Probes; D001224:Aspartic Acid; D000409:Alanine; D005998:Glycine