Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-03
2002-05-14
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007500, C435S007900, C435S007950, C435S018000, C435S028000, C436S501000, C536S023100, C536S024300
Reexamination Certificate
active
06387618
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to an assay method and composition for determining genetic toxicity. More particularly, the invention relates to a whole genomic DNA probe and to a method for using the DNA probe in a micronucleus assay.
2. Description of the Related Art
The mouse bone marrow micronucleus assay is used for the detection of damage to chromosomes or mitotic apparatus induced by particular compounds such as pharmaceutical drugs, environmental chemical agents, etc. (see, for example, Schmid, W. “The Micronucleus Test”, Mutation Res. 31, 9 (1975 ), Salamone, et al.,“Towards an Improved Micronucleus Test: Studies on 3 Model Agents, Mitomycin C, Cyclophosphamide and Dimethylbenzanthracene:” Mutation Res. 74, 347 (1980), and Heddle, J. A., et al. “The Induction of Micronuclei as a Measure of Genotoxicity: A Report of the U.S. Environment Protection Agency Gene-Tox Program, Mutation Res. 123, 61 (1983), Salamone, M. F. all incorporated herein by reference). The test is based on the observation that mitotic cells with chromatid breaks or chromatid exchanges exhibit disturbances in the anaphase distribution of their chromatin. After telophase, this displaced chromatin can be excluded from the nuclei of the daughter cells and is found in the cytoplasm as a micronucleus.
In the conventional mouse bone marrow micronucleus assay, mice are exposed to a particular test substance, and then bone marrow cells of the exposed animals are isolated. The isolated cells are immediately smeared onto a slide, stained with a reagent and examined under a microscope for the presence of micronuclei. Giemsa stain has traditionally been used as the staining reagent; however, because of the tendency of Giemsa stain to stain artifacts that resemble micronuclei, DNA-specific fluorescent stains such as acridine orange are now being used. See, for example, Hayashi, M., Sofuni, T. and Ishidate, M. Jr. 1983, “An application of acridine orange fluorescent staining to the micronucleus test”. Mutat. Research, 120:241-247; Hayashi, M., Morita, T., Kodama, Y., Sofuni, T. and Ishidate, M. Jr. 1990, “The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides” Mutat. Research, 245:245-249 and MacGregor, J. T., Wehr, C. M. and Langlois, R. G. 1983, “A simple fluorescent staining procedure for micronucleus and RNA in erythrocytes using Hoechst 33258 and pyronin Y” Mutat. Research, 120:269-275, all incorporated herein by reference.
DNA-specific fluorescent stains used in the mouse bone marrow micronucleus assay have several disadvantages that limit their usefulness. First, fluorescent microscopy requires expensive, specialized fluorescent microscopes equipped with special multi-filter cube switching and low light level video hardware. Fluorescent microscopes require a high degree of technical sophistication to use effectively. Second, fluorescent-stained slides fade over time and therefore cannot be stored and archived for future reference. Third, color signatures achieved by fluorescent detection may be unstable due to differential fluorescent bleaching rates. Fourth, automation of the finding and scoring of fluorescent stained micronuclei is costly and difficult. Finally, fluorescent preparations may cause swelling of chromosomes and loss of fine detail.
Thus, there is a need for a method for detecting micronuclei that allows the use of standard light field microscopy equipment and does not require expensive, specialized fluorescent microscopy equipment. Moreover, there is a need for a method of detecting micronuclei that provides a permanent record of the assay that can be stored and archived. Moreover, there is a need for a method of detecting micronuclei that produces a stable color signature. Moreover, there is a need for a method of detecting micronuclei wherein the finding and scoring of micronuclei can be easily automated. Moreover, there is a need for a method of detecting micronuclei that does not cause the swelling of chromosomes.
SUMMARY OF THE INVENTION
It has now been found that micronuclei in cells may be detected by means of a whole genomic DNA probe coupled with immunoenzymatic color pigment detection.
In particular, the invention provides a method for detecting the presence of micronuclei in cells of an organism. According to the method of the invention, cells of an organism are isolated and exposed to a hybridization probe of digested labeled, whole genomic DNA. The hybridization probe is labeled with a first binding member that allows it to bind specifically to a second binding member. The hybridization probe hybridizes with DNA in the cells, including DNA contained in micronuclei, if present. The hybridization probe is detected by exposing the cells to a compound comprising the second binding member coupled to an enzyme capable of reacting with a chromogenic substrate to convert the chromogenic substrate into a colored pigment. The compound binds to the first binding member. When the cells are exposed to a chromogenic substrate, the chromogenic substrate is converted into a colored pigment, thereby indicating the presence and location of DNA in the cells. The presence of micronuclei is indicated by the presence of colored pigment outside the nucleus of the cells. The cells are then examined and scored for the presence or absence of micronuclei.
In another aspect, the invention provides a hybridization probe for detecting micronuclei in mouse cells, the hybridization probe being made by digesting whole genomic mouse DNA into DNA fragments and then labeling the DNA fragments.
In another aspect, the invention provides a test kit for assaying cells for the presence of micronuclei, the test kit comprising
(a) a hybridization probe, the hybridization probe comprising digested, labeled whole genomic DNA, the digested genomic DNA being labeled with a first binding member capable of specifically binding with a second binding member,
(b) a compound comprising the second binding member coupled to an enzyme capable of reacting with a chromogenic substrate to convert the chromogenic substrate into a colored pigment, and
(c) a chromogenic substrate.
The invention overcomes the above-described disadvantages of the conventional fluorescent staining in a micronucleus assay. The use of a DNA-specific whole genomic DNA hybridization probe minimizes the staining of cell artifacts and thereby minimizes false positive results in the micronucleus assay. The colored pigment used in the detection of micronuclei can be visualized using standard light field microscopy, and expensive specialized fluorescent microscopes is not needed. Brightfield illumination may be used instead of epi-illumination (which is required to excite flurescence preparations and which is more uneven). The colored pigment used in the invention allows for permanent staining of cells, which allows for assay slides to be stored, archived and reanalyzed for years. The color signatures used in color pigment detection according to the present invention are more stable than fluorescent preparations and there is less variation in intensity of labeled elements within fields of view and between fields. The automation of finding and scoring of micronuclei is more easily achieved. The method of hybridization and color pigment staining of the present invention is less likely to cause swelling of chromosomes and loss of fine detail than methods of fluorescent staining.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Micronuclei may form in cells of an animal that is exposed to a genetically toxic substance or environment. The micronucleus assay, which involves exposing an animal to a particular substance or environment, then isolating cells from the animal and examining them for the presence of micronuclei, is a method for screening drugs and chemical agents to determine their genetic toxicity. See, for example, U.S. Pat. No. 5,229,265 to Tomesko; Parton, J. W., Probst, G. S. and Garriot, M. L. 1988, “The in vivo effect of 2,6-xylidine on induction of micronuclei in mou
Kolanko Christopher J.
Pyle Mark D.
Karasek John J.
Ketner Philip E.
The United States of America as represented by the Secretary of
Zitomer Stephanie W.
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