Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1990-04-12
1992-03-24
Wax, Robert A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
4352523, 4353201, 436501, 536 26, 536 27, 536 28, 935 77, 935 78, C12Q 168, C12N 120, G01N 33566, C07H 1512
Patent
active
050988239
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to nucleic acid fragments and their use in the diagnosis and prognosis of adenocarcinoma especially Familial Adenomatous Polyposis.
BACKGROUND OF THE INVENTION
Colorectal cancer is the second most common cancer in the United Kingdom and other developed countries in the West. Although usually not familial there is a rare dominantly inherited susceptibility to colon cancer, Familial Adenomatous Polyposis (FAP or Familial Polyposis Coli). During adolescence affected individuals develop from a few hundred to over a thousand adenomatous polyps in their large bowel. These have a sufficiently high probability of giving rise to adenocarcinomas that prophylactic removal of the colon is recommended in diagnosed FAP individuals. Polyps may also occur elsewhere in the gastrointestinal tract and the condition is often associated with other extracolonic lesions, such as epidermoid cysts, jaw osteomata and fibrous desmoid tumours. .sup.1,2,3,4
Adenomata have been suggested to be precancerous states for the majority of colorectal tumours .sup.5,6. Knudson.sup.7 has suggested that in both familial and non-familial cancers, dominant genes give rise to cancer susceptibility. He further proposed that these mutations act recessively at the cellular level, and that both copies of the gene must be lost for the cancer to develop. In sporadic cases both events occur somatically while in dominant familial cases susceptibility is inherited through a germ line mutation and the cancer develops following a somatic change in the homologous allele. This model was first substantiated by the elegant molecular work on retinoblastoma .sup.2a. Following the linkage of the disorder to chromosome 13.sup.3a, Cavenee et al.sup.2a, using polymorphic DNA markers for this chromosome, showed loss of heterozygosity in tumour material compared to normal tissue from the same patient, in both familial and non-familial forms. Similar results have now been obtained, following cytogenetic evidence which suggested the localisation of the disease on a specific chromosome, for Wilm's tumours .sup.4a-7a, acoustic neuromas.sup.8a as well as several other tumours .sup.9a,10a.
DISCLOSURE OF THE INVENTION
It has now been discovered that a DNA probe designated C11p11 localises to the chromosomal region 5q21-q22 and that this marker is closely linked to the disease gene for FAP. Moreover 20 to 25% of sporadic cases of adenocarcinoma of the colon involve changes in the human chromosome 5.
Accordingly the present invention provides a nucleic acid fragment capable of selectively hydridizing with the human chromosome 5 at the chromosomal region 5q20-q23.
In another embodiment, the present invention relates to a double stranded DNA fragment capable of selectively hybridizing under high stringency conditions with the human chromosome 5 at the chromosomal region 5q20-q23.
In other embodiments, the invention comprises processes for producing the subject fragments, nucleic acid probes comprising the fragments carrying a detectable label, processes for presymptomatic screening for FAP using these probes and processes for pathological classification of colonic tumors and precancerous polyps using the subject probes.
BRIEF DESCRIPTION OF THE FIGURES
Figure Legends
FIG. 1--Family 79 demonstrating segregation of FAP with a 3.9 Kb fragment using the probe C11p11.
Methods
Blood samples were collected from family members and lymphocytes separated and frozen for future transformation with EBV.sup.9. High molecular weight DNA was extracted from both the residue of the separation and from the transformed lymphocytes. The cells were lysed in a solution containing 0.33M sucrose, 5 mM MgCl, 10 mM Tris-HCl pH 7.5, 1% Triton X-100. Nuclei were pelleted at 8000 g at 4.degree. C. for 10 mins. and resuspended in 75 mM NaCl, 25 mM EDTA, 100 .mu.g/ml Proteinase K and 0.5% SDS. The mixture was incubated overnight at 37.degree. C. The aqueous phase was extracted 3 times in 2 mM Tris:2 mM EDTA buffered phenol and once in isoamyl alcohol/chlo
REFERENCES:
Nature, vol. 328, 13 Aug. 1987, W. F. Bodmer et al., pp. 614-616.
Nature, vol. 328, 13 Aug. 1987, E. Solomon et al., pp. 616-619.
Science, vol. 238, 4 Dec. 1987, M. Leppert et al., pp. 1411-1413.
Biological Abstracts/RRM, vol. 35, No. 64125, K. P. Meera et al. (Switzerland) 1987, vol. 46, No. 1-4, p. 661.
Biological Abstracts /RRM, vol. 35, No. 64065, M. Leppert et al. (Switzerland) 1987, vol. 46, No. 1-4, p. 647.
Genetic Analysis of Familial Polyposis Coli, Analysis of Gene Expression of Oncogenes in Colon Tumors of FPC by Kenji Sugio, Midical Inst. of Bioregulation, Kyushu University, Fukuoka 812, Japan, pp. 185-197.
The Gene for Familial Polyposis Coli Maps to the Long Arm of Chromosome 5, by M. Leppert et al., Science, vol. 238, No. 4832, pp. 1411-1413.
Bailey Carolyn J.
Bodmer Walter F.
Murday Victoria A.
Williamson Robert
Imperial Cancer Research Technology Ltd.
Wax Robert A.
Zitomer Stephanie W.
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