Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-08-22
2000-07-18
Houtteman, Scott W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
536 2431, 536 2432, C12Q 168
Patent
active
060905468
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the detection of mutations in Ras oncogenes, and in particular in the K-ras gene.
Ras oncogenes are implicated in the development of a range of cancers. In particular somatically induced, activating mutations at defined positions in ras genes are believed to be important causative events in the process of tumorigenesis. Ras gene mutations occur in approximately 30% of human tumours, including cancer of the lung, thyroid, colon, rectum, pancreas and breast, and certain melanomas and leukaemias, although their incidence does vary according to tumour type. In addition, certain experimental tumour systems have been shown to be associated with activated ras genes. More specifically, mutations of the K-ras gene have been reported to be as high as 90% in carcinomas of the pancreas, 50% in adenocarcinomas of the lung and 40% in adenocarcinomas of the colon.
Activation of the ras oncogenes appears to be most frequently associated with mutations at position 12, although activation at other positions e.g. positions 13 and 61 is also commonly observed. Thus, in recent years a number of tests for the detection of ras mutations have been developed as a means towards clarifying their functional role in tumorigenic pathways, and indeed for their potential utility in the diagnosis and prognosis of cancer.
Primer-mediated restriction fragment length polymorphism (RFLP) analysis was developed as a rapid, non-radioactive method for fast and simple large-scale detection of mutant ras genes (Kahn et al., 1990, Amplifications, 4, 22-26). This method relies upon the polymerase chain reaction (PCR) for amplification of the ras sequences and upon the introduction of restriction endonuclease sites permitting selective restriction cleavage of normal (wild type) ras sequences only, and not of mutant ras sequences. The ras gene primers used for PCR amplification in this process incorporate strategic nucleotide substitutions which serve to create restriction sites overlapping potential activating positions, e.g. positions 12, 13 or 61. The diagnostic restriction site of the target codon is lost in the presence of an activating mutation, thereby permitting detection of the mutant sequences.
However, the detection of ras mutations by this procedure is limited because both mutant and normal sequences are amplified by the PCR step, thereby compromising sensitivity (the number of mutant ras sequences, amongst normal, wild type, DNA may be very low). In more recent years therefore, modifications of the RFLP technique have been developed, whereby mutant ras sequences are selectively amplified (see for example Kahn et al., 1991, Oncogene, 6 1079-1083). This greatly enhances the diagnostic capability of the technique and enables the detection of mutant ras alleles in the presence of up to 10.sup.4 wild type sequences.
Nonetheless, although the combination of primer-mediated RFLP analysis with selective amplification of mutant sequences permitted a significant advance in ras mutation detection, there is still room for improvement, for example in the sensitivity and specificity of the method, and such improvements are continually being sought. There is therefore a continuing need for improved methods of detecting mutations in ras genes and the present invention is directed towards providing such an improved method.
In particular, we have directed our efforts towards the K-ras gene, and more specifically to the detection of activating mutations at codons 12, 13 and 61 of the K-ras gene, which are believed to be implicated in the development of many common cancers, most notably colo-rectal cancer. Thus, a novel and advantageous primer system has been designed, for use in amplification or RFLP-based ras mutation detection methods, which permit the detection of mutations at codons 12 and/or 13 of the K-ras gene. A further primer system permits detection of mutations at codon 61.
In one aspect, therefore, the present invention provides an oligonucleotide primer sequence for use in in vitro amplification, characterised i
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Breivik Jarle
Gaudernack Gustav
Houtteman Scott W.
Medinnova SF
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