Method for the quantitative assessment of global and specific dna repair capacities of at least one biological medium, and the applications thereof

Abstract

The invention relates to a method for the quantitative assessment of the global and specific DNA repair capacities of a biological medium, consisting in assessing the excision/resynthesis capacities of said medium, and to the applications thereof. The inventive method comprises the following steps: (a) a range of plasmids, each comprising distinct DNA lesions, is prepared; (b) the lesions present on each plasmid from the range of plasmids are characterised; (c) different plasmids from the range and at least one supercoiled control plasmid are deposited on a single solid support, using a pre-established configuration A 1 in order to form a functionalised support which is divided into different zones A 1 to A x , x corresponding to an integer which is equal to the number a of biological media to be tested simultaneously, each of zones A 1 to A x comprising said range of plasmids; (d) the functionalised support obtained in step (c) is incubated with different repair solutions; (e) said functionalised support is washed at least once; (f) the signal produced by the marker incorporated into the DNA during the repair reaction in step (d) is measured directly or indirectly; (g) the signal corresponding to each plasmid deposit is recorded and quantified; and (h) the ratio of the signals from plasmids comprising lesions in relation to the control plasmid deposited with same is determined.

1 - 21 . (canceled)  
   
   
       22 . A method for quantitative assessment of overall and specific DNA repair capacities of at least one biological medium, which method comprises the following steps of: 
 a) preparing a range of plasmids, each comprising distinct DNA lesions, by independent treatment of said plasmids with at least one physical or chemical treatment means or both and recovering a supercoiled fraction of each of said plasmids,    b) characterizing the lesions present on each of the plasmids of said range of plasmids,    c) depositing the plasmids of said range of plasmids, and at least one supercoiled control plasmid without lesions, onto a single solid support, according to a pre-established configuration A, so as to form a functionalized support divided into different zones A 1  to A x , x corresponding to an integer equal to the number of biological media to be simultaneously tested, each zone A 1  to A x  comprising said range of plasmids,    d) incubating said functionalized support obtained in step (c) with various repair solutions, each comprising at least one biological medium that may contain enzyme activities for repair, ATP, an ATP-regenerating system, a labeled nucleotide triphosphate and any other component necessary for the activity of the repair enzymes present in said biological medium, each of said repair solutions being deposited, prior to said incubation, in each of said different and pre-established zones A 1  to A x  of said functionalized support,    e) washing said functionalized support at least once,    f) directly or indirectly measuring the signal produced by the label incorporated into the DNA during the repair reaction in step (d), in each of said different and pre-established zones A 1  to A x ,    g) recording and quantifying the signal corresponding to each deposit of plasmid in each zone A 1  to A x , and    h) determining the ratio of the signals of the plasmids comprising the lesions relative to the control plasmid jointly deposited.    
   
   
       23 . The method of  claim 22 , wherein the plasmids according to step (a) have a double-stranded supercoiled form.  
   
   
       24 . The method of  claim 22 , wherein in step (a) the at least one physical or chemical treatment means which induce a lesion of the DNA are selected from the group consisting of those that induce: the formation of a single lesion, the formation of a limited number of lesions and the formation of various lesions belonging to the same family.  
   
   
       25 . The method of  claim 22 , wherein, in step (a) multiple treatment means are used on each plasmid of said range of plasmids.  
   
   
       26 . The method of  claim 22 , wherein, in step (b) the characterizing of the lesions comprises (i) taking a fraction of each plasmid with lesions, (ii) digesting each of said fractions with enzymes that release the nucleosides from the DNA, and then (iii) analyzing the result of the digestion using a combination of separative techniques coupled to a quantitative analytical technique.  
   
   
       27 . The method of  claim 26 , wherein the digestion is carried out using at least one of the following enzymes: calf spleen phosphodiesterase, P1 nuclease, snake venom phosphodiesterase, or alkaline phosphatase.  
   
   
       28 . The method of  claim 26 , wherein the result of the enzyme digestion is analyzed by means of one of the following techniques: high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry, by HPLC coupled to gas chromatography or by HPLC coupled to electrochemical detection.  
   
   
       29 . The method of  claim 22 , wherein, prior to step (c), the supercoiled forms of the plasmids obtained in step (a) are purified by sucrose gradient centrifugation or cesium chloride gradient centrifugation or both.  
   
   
       30 . The method of  claim 22 , wherein, also prior to step (c), each of the plasmids of the range of plasmids is diluted to a concentration of between 5 and 100 μg/ml, in a diluting buffer.  
   
   
       31 . The method of  claim 30 , wherein said buffer has a pH of between 6.5 and 8.0.  
   
   
       32 . The method of  claim 31 , wherein said buffer further optionally comprises a salt and a non-ionic surfactant.  
   
   
       33 . The method of  claim 22 , wherein, in step (c) the volumes of the deposits of the range of plasmids are between about 100 and 1000 picoliters.  
   
   
       34 . The method of  claim 22 , wherein, in step (c), said support is a support that has been sensitized so as to increase its affinity for the DNA, and is an organic or inorganic material selected from the group consisting of glass, silicon and compounds thereof, and synthetic or nonsynthetic polymers, and the surface of which is optionally functionalized.  
   
   
       35 . The method of  claim 34 , wherein said support consists of glass slides coated with poly-L-lysine that adsorb the DNA, or glass slides functionalized with epoxy groups that form covalent bonds with the DNA.  
   
   
       36 . The method of  claim 34 , wherein said support comprises different zones A 1  to A x , each of said zones comprising: 
 a) at least one deposit of control plasmid, and    b) a deposit of plasmid containing photoproducts, and/or    c) a deposit of plasmid containing oxidative damage, and/or    d) a deposit of plasmid containing etheno-bases, and/or    e) a deposit of plasmid containing DNA breakages, and/or    f) a deposit of plasmid containing carcinogenic substance adducts.    
   
   
       37 . The method of  claim 22 , wherein in step e), the support is washed at least once with a saline solution containing a nonionic surfactant, in particular a 10 mM phosphate buffer containing Tween 20, and is then subsequently rinsed with water at least once.  
   
   
       38 . The method of  claim 22 , wherein in step f), the signal is measured by means of a method suitable for the label.  
   
   
       39 . The method of  claim 22 , wherein, in step (g), said signals are quantified using a device capable of exciting the label and of measuring the signal emitted subsequent to the excitation.  
   
   
       40 . The method of  claim 39 , wherein, in step (h) of the method, a numerical ratio of the signals obtained with the plasmids containing the lesions to the signal obtained with the control plasmid located on the same support is established.  
   
   
       41 . The method of  claim 22 , wherein the quantitative assessment is for establishing a repair profile of the at least one biological medium.  
   
   
       42 . The method of  claim 22 , wherein the quantitative assessment is for diagnosing a repair-related disease.  
   
   
       43 . The method of  claim 22 , wherein the quantitative assessment is for assessing the influence of a physical or chemical treatment means on repair capacities of the at least one biological medium.  
   
   
       44 . The method of  claim 22 , wherein the quantitative assessment is for screening substances capable of modulating a repair system of the at least one biological medium.  
   
   
       45 . The method of  claim 22 , wherein in step d), said incubating is effected at a temperature of about 30° C. for about 1 to 5 hours.  
   
   
       46 . The method of  claim 45 , wherein said incubating is for about 3 hours.