Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1997-11-25
2001-07-17
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S091100, C435S440000, C435S325000, C514S04400A, C536S022100, C536S023100, C536S023200, C536S023400, C536S023500
Reexamination Certificate
active
06261767
ABSTRACT:
The present invention has for its object a process for the qualitative and quantitative detection of damage to desoxyribonucleic acid due to physical or chemical agents as well as a process for the qualitative and quantitative detection of the activity of modulatory substances for the repair of cellular extracts of damaged DNA.
In the following description and claims, there is referred to as a “damaging product”, any pure specific chemical agent, any artificial mixture of chemical agents, or any natural composition of chemical agents or even physical agents such as radiation, particularly ionizing and ultraviolet radiations.
It is known that DNA serve to support the genetic information and that it can be damaged by damaging products with important consequences if the damage is not repaired. Thus, the cellular division is dependent on this information and even the life of the cell is concerned because the genes expressed in the cell are necessary for good cellular functioning.
Also, damage can lead to a mutation of DNA, which can give rise to dysfunction of the cell and as a result introduce a problem into the organism.
It is therefore important to analyze the DNA reactions of an individual as to damaging products so as to get pertinent information, particularly to follow the evolution of certain maladies or for preventing the consequences of them. It is therefore necessary, from cells contained in the blood or in some tissue, to recover DNA then to purify and analyze its behavior with regard to damaging products by determining the consequences induced by this damage.
Thus, in the medical industry, it can be needed to determine qualitatively and quantitatively the genotoxicity or the anti-genotoxicity of a compound or mixture of compounds as to DNA, and if possible the capacities for repairing the damage thus generated. Such a determination is useful as to cells developed and cultivated in vitro or isolated ex vivo. Applications of this type of determination are the detection of genotoxic xerobiotics, following patients in chemotherapy, or following persons working in contact with genotoxic substances.
Another object of the process according to the invention is, in the case of damaging chemical agents, to seek to verify the inhibitory capacities of a compound as to this product, which is particularly the case for oxidants and antioxidants.
There are known different tests concerning DNA and particularly that of patent application EP 0 472 482 in the name of the present applicant, which relates to the dosage with microquantities of DNA present in an extracellular position in a biological liquid, particularly in blood plasma.
This process consists in fixing by adsorption the DNA on a solid support thanks to substances having a high affinity for DNA. Thus, this substance can be chosen particularly from cationic macromolecular complexes.
The solid support is generally a microtitration plate comprising wells, which are sensitized by incubation in a medium containing cationic macromolecular complexes.
This plate is incubated with the specimen whose DNA proportion it is sought to determine. The DNA is adsorbed and the plate is washed in borate buffer with a surfactant or a tris(hydroxymethyl)aminomethane buffer.
The DNA thus fixed is subjected to a molecular biological reaction using several specific enzymes of DNA so as to incorporate at least one nucleotide chemically marked with a substance that can subsequently be detected, for example by enzymatic reaction.
Such incorporation is provided in this process prior to detection by the “Nick translation” reaction, which is to say by a reaction of displacement of haplotomic cutting. Such a marker, described in this previous application, is a modified nucleotide marked with an avidine/enzyme compound or streptavidine/enzyme, the enzyme being peroxidase.
After interruption of the enzymatic reaction, the detection of the incorporated marker is carried out by means of an apparatus adapted to quantify the emitted signal. There is thus measured with a spectrophotometer the colored reaction obtained with biotine.
Such a process of determination of DNA present in very small quantity in a specimen has numerous advantages, particularly:
no extraction of DNA prior to its determination,
no need to use a “hot” marker, which is to say radioactive,
no cumbersome specific apparatus,
high sensitivity,
simultaneous use for more than one hundred specimens, and
obtention of results in several hours.
Such a process therefore has a well determined object which is the determination of DNA so as to determine possible variations of the quantity of DNA, particularly in humans but also under experimental conditions to quantify the phenomena of cellular death.
There is known a test described by Wood et al. [(1988) Cell, 53, 97-106] and Sancar et al. [Sibghat-Ullah et al. (1989) Nucleic Acids Res., 17, 4471-4484], which permits detecting damage to DNA.
This test consists in placing two very pure plasmids of different weights, the one damaged and the other undamaged serving as a reference, in the presence of a cellular extract, transcriptionally active, in the presence among other things of a known composition of dNTP, of marked DATP and of magnesium.
The damage is detected, cut out, and the subsequent reparative synthesis of DNA permits incorporation of radio-marked dNTP whose presence will then be quantified.
A certain percentage of damage is repaired by this incision-excision mechanism and resynthesis, of the order of 7%.
The plasmids are linearized, and caused to migrate on an electrophoresis gel so as to ensure the separation and the radioactive signal emitted by the repaired chains is studied, in which a “hot” marker is present. The signal ratio gives the repair ratio.
The incision-excision activity can also be directly measured [Calsou and Salles (1994) Biochem. Biophys. Res. Comm., 202, 788-795].
Such a test has numerous drawbacks and particularly the fact that the application in industry under satisfactory automated conditions is difficult so that this test is only for laboratories. The delay of obtaining the results, (of the order of two days), is too long and the cost too high. As another drawback, can be cited the requirement to use purified plasmids, of quantities of the order of 200-300 ng and on this subject it should be noted that the test does not seem to be transposable to the analysis of DNA damage in all in vitro or ex vivo sources.
Moreover, it is noted that the markers are isotopes whose manipulation is less easy and that the replacement by markers of another kind is difficult.
Furthermore, in the field of markers, there is a strong tendency to consider that radioactive markers are more sensitive than the others so that researchers are instinctive- ly inclined to use such markers and even to be dissuaded from using cold markers.
Of course tests are known to detect the mutagens because a relationship exists between damage and mutagenesis. These are more particularly the AMES test [Ames et al. (1973) Proc. Natl. Acad. Sci. USA, 70, 2281-2285] complimented by a micronucleus test [Mac Gregor et al. (1987) Mutat. Res. 189, 103-112] which permits the analysis of chromosomic breaks and of tests permitting detecting mutations in genes such as HPRT coding for a biosynthesis enzyme based on DNA.
In the pharmaceutical industry, there is often used and above all in case of doubt about the preceding tests, the UDS test “Unscheduled DNA Synthesis” which consists also in measuring the incorporation of one or several marked nucleotides into DNA of cells subjected to potentially genotoxic agents.
This test has another drawback of involving the manipulation of radio-isotopes and being semi-quantitative.
An improvement of UDS proposed a non-radioactive marker [Selden et al. (1994) Mutat. Res., 315, 147-167], but this is less interesting because the analysis of the results requires the use of flow cytometry, which is a very difficult technique.
The process according to the invention therefore has for its o
Calsou Patrick
Fournie Gilbert
Provot Christian
Salles Bernard
Riley Jezia
Societe Francaise de Recherches et d'Investissements (SFRI)
Young & Thompson
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