Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-14
2002-09-10
Jones, W. Gary (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S287200, C435S288400, C435S288700, C435S325000, C435S810000, C536S024310, C536S024330, C536S025400, C424S489000, C502S416000
Reexamination Certificate
active
06448002
ABSTRACT:
This application is the national phase of PCT/DE97/01894, filed Aug. 26, 1997.
BACKGROUND OF THE INVENTION
The invention relates to a method for detecting clinically relevant mutations in the DNA sequence of the ki-ras oncogene in DNA, preferably in stool DNA, its use, as well as a test kit for the early diagnosis of tumors, particularly of tumors of the pancreas and the large intestine.
Worldwide, tumors of the large intestine and pancreas are the most frequently occurring cancerous affections and are in third or fourth place in the mortality statistics of malignant growths. Problems of these diseases come to the fore particularly clearly in the case of pancreatic cancer. Because the disease is asymptomatic for a long time, pancreas carcinomas are diagnosed so late, that the average five-year survival rate of patients does not exceed 2% in spite of flawless surgical techniques.
At the present time, there are no satisfactory laboratory parameters for the early diagnosis of pancreatic tumors. For the diagnosis of colorectal carcinoma, the occult blood test is used, for which occult blood is detected in the stool. The analytical reliability of this test is not satisfactory, because occult blood occurs in the stool even in the case of non-malignant diseases, such as hemorrhoids and because positive findings can furthermore also be caused by a large number of interfering stool components, such as peroxidases and catalyses. On the other hand, it is well known that tumors with a diameter of less than 2 cm do not give off sufficient blood to be noticed in an occult blood test method. Even in the case of advanced tumors of the large intestine, the diagnostic sensitivity of the occult blood test is only 50 to 70%.
One possible alternative for the early diagnosis of tumors of the pancreas and the large intestine is offered by the molecular biological detection of relevant mutations in oncogenes, preferably in the ki-ras proto-oncogene. For example, it was possible to show that mutations in the ki-ras proto-oncogene occur in 75 to 90% of pancreatic carcinoma (Almoguera, S., Shiate, D., Forrester, K., Martin, J., Arrheim, N., Perucho, M. (1988) Cell 53, 549-554) as well as in the case of at least 50% of colorectal carcinoma (Forrester, K., Alomugera, C., Han, K., Grizzle, W. E., Perrucho, M. (1987) Nature 327, 298-303).
Until now, the frequency, with which an oncogene has been found for a specific tumor, has been highest in the case of pancreatic carcinoma. Moreover, the relevant mutations are limited to the codons of three amino acids and, in the case of pancreatic carcinoma, to only one amino acid. This fact can simplify the detection of mutations appreciably.
In the case of tumors of the large intestine, as well as in the case of pancreatic tumors, the possibility exists of a non-invasive detection of the oncogene status in the stool. Such a detection was shown for the first time in 1992 by Sidransky and fellow workers (Sidransky, D., Tokino, T., Hamilton, S. R., Kinler, K. W., Levin, B., Vogelstein, B. (1992); Science 256, 102-105). The detection is successful because, on the one hand, a sufficient number of epithelial cells of the pancreas and the intestine reach the stool, tumor cells possibly being more stable than normal epithelial cells under the conditions existing in the stool. On the other, bacteria have no gene for ki-ras, so that the diagnosis is not affected by interference of intestinal flora and epithelial cells. Adenoma, up to a size of 1 cc, can be diagnosed by way of the detection of activated ki-ras.
In spite of these advantageous pre-conditions for a laboratory diagnostic application, there is, up to now, no method for detecting ki-ras mutations in stool DNA, which can be used routinely.
The main problem here lies in the enormous difficulty of isolating DNA of adequate quality from stool samples at a justifiable expense. The extraction method, practiced most frequently at the present time, contains a series of purification steps requiring several hours and extends in total to at least one working week.
Further improvements by Caldas and co-workers resulted in a method, which also required several days for its implementation (Caldas, C., Hahn, S. A., Hruban, R. H., Redston, M. S., Yeo, C. J., Kern, S. E. (1994); Cancer Res. 54, 3568-3573).
Stool is a complex mixtures of cells, which have flaked off, microorganisms, undigested food components, mucous materials and coloring matter, as well as of other soluble and insoluble components of the gastrointestinal tract. Such a complex composition causes a large number of inhibitory materials to be present, which are contaminating components of the isolated DNA solution and, as well, are also intercalated directly in the DNA or bound to the DNA and, for this reason, prevent the use of the isolated DNA for further investigations.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to develop a method of detection for a corresponding gene diagnosis system, which can be used routinely, a suitable method for extracting DNA having a key function for establishing a routinely useful ki-ras test, which can also be automated, as well as to make available a test kit based on the method.
This objective is realized in accordance with claims
1
to
15
. Pursuant to the invention, it is accomplished owing to the fact that genomic DNA is extracted by means of multiple purification steps from material samples, preferably from stool samples, all inhibitory materials being eliminated in a highly selective manner, so that the isolated genomic DNA is available without problems for further applications.
Starting materials, within the sense of the invention, are stool samples, biopsy samples from gastrointestinal polyps after endoscopic removal, ercp fluids from the pancreas, sputum and optionally also blood, plasma, serum and urine.
Especially stool samples are incubated pursuant to the invention in a first step with materials, which have absorbing properties, for removing inhibitory materials. The cells, contained in the samples, are lysed with a buffer, which contains chaotropic salts. The genomic DNA, which is subsequently bound to a mineral carrier material, is purified further in a further washing process and then dissolved off from the carrier material by means of a buffer of low ionic strength. In a subsequent purification step, the material or materials (inhibitors), bound in or to the DNA, are then also removed. Especially these materials, bound in or to the DNA, represent potent inhibitors. These are displaced from the DNA by incubation of the already isolated DNA with a buffer, which has an ionic strength greater than 4M and contains a chaotropic salt, such as sodium iodide. A subsequent addition of the carrier material for binding the DNA once again, followed by washing and elution of the DNA, form the conclusion of the purification process. The DNA, isolated in this manner from the samples, is now available for further molecular biological diagnostic techniques.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In particular, the following purification steps are carried our pursuant to the invention:
a) optionally incubating with preferably chromatographic materials with absorption properties, preferably with a solution of activated charcoal, for the removal of inhibitory materials when a stool sample is used;
b) lysis of the cells, contained in the material samples, with a buffer, which contains chaotropic salts, such as guanidine isothiocyanate, guanidine hydrochloride, lithium chloride or lithium chloride/urea mixtures with an ionic strength greater than 4M;
c) incubation of the lysate with a mineral carrier material for binding the DNA; the carrier material preferably is a highly disperse, non-porous silica with a particle size of 7 nm to 1 &mgr;m and preferably of 40 nm and with a specific surface area of 10 to 300 m
2
/g and preferably of 50 m
2
/g;
d) separation of the carrier material from the lysate by centrifugation;
e) washing the DNA, bound to the carrier material, preferably with a buffer consisting of 50 mM NaCl
Bendzko Peter
Berndt Hans-Christoph
Hillebrand Timo
Invitek GmbH
Johannsen Diana
Jones W. Gary
Norris McLaughlin & Marcus P.A.
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