Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-19
2001-10-30
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S183000, C424S178100
Reexamination Certificate
active
06309838
ABSTRACT:
The subject of the present invention is a method for the qualitative and quantitative detection of impairments of DeoxyriboNucleic Acid (DNA), it being possible for these impairments to be due to the metabolism of the cell or to endogenous or exogenous, physical or chemical agents, as well as a method for the qualitative and quantitative detection of the ligand(s) recognizing impaired DNA.
The most satisfactory definition (among the many which are much debated) of genotoxicity is a general definition which envisages the appearance of physical or chemical impairments of the DNA due to a direct action of the genotoxic agent or of a metabolite, as well as its biological consequences (Butterworth, 1990, Mutat. Res., 239, 117-132; Ashby, 1992, Mechanism of Carcinogenesis in risk identification, Vainio H., Magee P. N., McGregor D. B. & McMichael A. J. (eds) IARC, Lyon, 135-164). Among the genotoxic agents, many molecules or physical agents are capable of inducing the appearance of mutations which are detected by various bacterial or eukaryotic systems. The system for detecting mutagens which is most generally used is that described by B. N. Ames (Ames et al., 1973, Proc. Natl. Acad. Sci. USA, 70, 2281-2285), supplemented by the so-called micronucleus test (Mac Grégor et al., 1987, Mutat. Res., 189, 103-112).
Since mutagenesis appears to be in numerous cases the consequence of the presence of lesions (due in particular to genotoxic agents), many systemes capable of detecting and quantifying these types of damage to DNA have been developed. The detection of DNA lesions involves physicochemical techniques such as post-labeling (Randerath et al., 1981, Proc. Natl., Acad. Sci. USA, 78, 6126-6129), alkaline or neutral elution (Kohn et al., 1976, Biochemistry, 15, 4629-4637), immunological techniques (Philips D. H., Chemical-carcinogenesis and mutagenesis, vol. I, 503-546, Springer Verlag, Heidelberg, 1990, Cooper and Grover (eds), or calls into play the consequences of events for the repair of these lesions such as the comet assay (Singh et al., 1998, Exp. Cell Res., 175, 184-191).
Some assays use the lesion-repair capacity of the cell whose mechanism involves a step of synthesis of DNA de novo. This DNA polymerization step may be quantified using radiolabeled nucleotides (Cleaver, 1984, Methods for studying excision-repair of eukaryotic DNA damaged by physical and chemical mutagens (Kilbey B. J., Nichols W. & Ramel C., eds) 33-69, Elsevier, Amsterdam). This assay has been called UDS (Unscheduled DNA Synthesis) and has been used for the detection of a genotoxic agent or for the evaluation of the repair capacities of cells. This UDS test has been improved in the sense of the non-use of a radioactive marker (Selden et al., 1994, Mutation Res., 315, 147-167) but this improvement is made relative because of the use of flow cytometry which makes the method cumbersome.
The repair of DNA is mainly due to the system for the excision of lesions, which system has been recently reproduced with cellular extracts (Wood et al., 1988, Cell, 53, 97-106; Sibghat-Ullah et al., 1989, Nucleic Acids Res., 17, 4471-4484). This assay uses treated and untreated (control) plasmids incubated in the presence of transcriptionally active extracts (Manley et al., 1983, Meth. Enzymol., 101, 568-582). The repair reaction consists in the incision-excision of the lesions and then the resynthesis of a DNA fragment. The method takes advantage of this step during which one or more radiolabeled nucleotides are incorporated. Two major mechanisms are involved in the restoration of the damage to DNA: the excision of nucleotides (NER) and the excision of bases (BER). These two mechanisms will be described later.
In the case of this test in vitro, the repair of lesions by NER involves only a small percentage of lesions, of the order of 7%.
These assays are not applicable to a large screening or, in a wider sense, industrial research application for various reasons:
(i) the number of samples which can be treated simultaneously (of the order of a few tens) is too low,
(ii) the time required for the analysis (about 2 days) is relatively long,
(iii) the use of radiolabeled molecules restricts this test to approved laboratories, (iv) the test is restricted to the use of highly purified plasmid DNA (supercoiled form).
Taking these constraints into account, a new in vitro repair-synthesis assay capable of overcoming the disadvantages of the other systems has been developed, by allowing the simultaneous analysis of more than 100 samples in 5 hours, with a nonradioactive detection system. This method, which also allows the use of DNA of different types, has been the subject of a French patent application No. 95 03230 of Mar. 15, 1995 and of a PCT application No. PCT/FR 96/00391 of Mar. 13, 1996, published under the No. WO 96 28571 on Sep. 19, 1996.
The object of other fairly similar techniques is the direct detection of damaged DNA, but none relates to the detection of the repair systems linked to lesioned DNA. For example, the quantitative detection of the impairments of DNA by incorporation of radioactively labeled nucleotides is described in patent applications Nos. WO 96 24688 and WO 96 23895 and in the following articles: Salles B. et al. In vitro eukaryotic DNA excision repair assays; an overview; Biochimie, vol. 77, No. 10, 1995; Calsou P. and Salles B. Measurement of Damage-specific DNA incision by nucleotide excision repair in vitro; Biochemical and Biophysical Research Communications, vol. 202, No. 2, Jul. 29, 1994; Calsou P. and Salles B. Properties of damage-dependent DNA incision by nucleotide excision repair in human cell-free extracts, Nucleic Acids Research, vol. 22, No. 23, 1994; Salles B. and Calsou P. Rapid quantification of DNA repair synthesis in cell extracts, Analytical Biochemistry, vol. 215, 304-306, 1993.
The patent application No. WO 96 24688 relates in particular to the use of anti-modified DNA antibodies and the article by Salles B. et al. (A chemiluminescent microplate assay to detect DNA damage induced by genotoxic treatments, Analytical Biochemistry, vol. 232, 37-42, 1995) relates to the incorporation of DIG-11-dUTP and the use of anti-DIG-11-dUTP antibodies for the detection of lesions.
Among the prior art documents, none describes or suggests the present invention as defined below.
DESCRIPTION OF THE INVENTION
Thus, the subject of the present invention is a method for identifying an impairment of a DNA sequence which can allow a detection of all the impairments of DNA by means of a method whose use is relatively simple.
To do this, the present invention proposes a method for identifying an impairment of a DNA sequence, characterized in that:
a) said DNA sequence is brought into contact with a composition containing at least one compound recognizing the type of impairment implicated, called a ligand, in a medium allowing the recognition,
b) the recognition of the impairment by said ligand is revealed.
By recognition of the impairment by said ligand, it is understood that the direct or indirect attachment of the ligand to the damaged DNA is detected. This may be an indirect attachment when the ligand is part of a complex for example.
Impairment is understood to mean, first of all, the damage to DNA. This may be of physical origin (for example thermal, or ionizing or nonionizing radiation) or of chemical origin. These factors which damage DNA may be of exogenous or endogenous origin. The types of lesion generated on the DNA can be roughly classified into 5 types:
adducts (generally covalent formation of complex, but which may involve another type of chemical bonding such as the coordination bonding of a molecule to a base of the DNA),
coupling of bases (achieved by a supply of energy, the best known type being found in the dimers of pyrimidine bases formed under the action of UVC or UVB radiation),
complexing of bases of the DNA with proteins (this type of complexing, most often covalent, also being the consequence of a supply of energy),
oxidative damage causing modifications of the structure of the DNA (in
Chaubron Franck
Provot Christian
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Forman B J
Genolife
Myers Carla J.
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