EP0057553A1
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Method for the fluorescent detection of damaged DNA and kit for use therein
Assignee: Atomic Energy of Canada Ltd AECL
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Filing: 1982-01-26
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Granted: —
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Expires: 2002-01-26
Abstract
Abstract A sensitive method for detecting damage to DNA, e.g. resulting from exposure of living cells or whole organisms to low doses of radiation or chemicals, is described comprising: partial lysis of exposed cells to access the DNA; alkaline denaturing the DNA; after a selected interval stopping the denaturation by lowering the pH to a selected value; rendering the lysate homogeneous; adding an appropriate fluorescent dye which interacts preferentially with double-stranded DNA and measuring the resulting fluorescence. The decrease in fluorescence (compared to that before denaturation) is a measure of the rate of DNA denaturation which is directly proportional to the extent of DNA damage. Several factors, particularly the lowered pH at which the denaturation is stopped, have been found important for increased sensitivity. The method is suitable for monitoring the effect on DNA in living cells of environmental factors, drug and radiation therapy, and for toxicology studies. A kit adapted for carrying out the test method is described.
Inventors
- 👤 Hyman C. Birnboim
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 01:48
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/52; G01N2800/00; G01N2800/52; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
175 words
A method of measuring the extent of DNA damage in cells which have been exposed to an actual or potential damaging influence, comprising: (a) partially lysing the cells under test to render the DNA accessible; (b) incorporating an alkaline reagent, in sufficient amounts to denature DNA, into at least one sample comprising the cell lysate, and allowing DNA strand separation to proceed; (c) at a chosen point of time during the denaturation period, lowering the pH of each sample to a selected pH sufficiently low to stop the alkaline denaturation, but above that causing any precipitation; (d) providing that each sample is substantially homogeneous.; (e) adding a fluorescent dye which interacts and fluoresces at the lowered pH with non-denatured double strand DNA but not with single strand DNA or other cell components, to each sample of lowered pH and measuring the resulting fluorescence; and (f) comparing the fluorescence with that of known undamaged DNA subject to the same denaturation, the amount of any decrease in fluorescence being directly proportional to the extent of DNA damage.
Claim 2
Independent Claim
175 words
A method of measuring the extent of DNA damage in cells which have been exposed to an actual or potential damaging influence, comprising: (a) partially lysing the cells under test to render the DNA accessible; (b) incorporating an alkaline reagent, in sufficient amounts to denature DNA, into at least one sample comprising the cell lysate, and allowing DNA strand separation to proceed; (c) at a chosen point of time during the denaturation period, lowering the pH of each sample to a selected pH sufficiently low to stop the alkaline denaturation, but above that causing any precipitation; (d) providing that each sample is substantially homogeneous.; (e) adding a fluorescent dye which interacts and fluoresces at the lowered pH with non-denatured double strand DNA but not with single strand DNA or other cell components, to each sample of lowered pH and measuring the resulting fluorescence; and (f) comparing the fluorescence with that of known undamaged DNA subject to the same denaturation, the amount of any decrease in fluorescence being directly proportional to the extent of DNA damage.
Claim 3
Dependent Claim
17 words
A method according to claim 1, wherein the cells are partially lysed in the presence of urea.
Claim 4
Dependent Claim
29 words
A method according to claim 1 or 2, wherein the lysing of the cells is carried out with the cells suspended in a neutral buffer containing an osmotic stabilizer.
Claim 5
Dependent Claim
28 words
A method according to claim 3 wherein the cells are suspended in a phosphate buffer containing inositol and a source Mg or Ca ions as the osmotic stabilizer.
Claim 6
Independent Claim
31 words
A method according to any one of the preceding claims wherein the denaturation of the lysed cells is carried out using alkaline solutions of different densities to facilitate obtaining pH equilibrium.
Claim 7
Independent Claim
25 words
A method according to any one of the preceding claims wherein the alkaline denaturing step (b) takes place at a pH of 12 or more.
Claim 8
Independent Claim
25 words
A method according to any one of the preceding claims, wherein the denaturing is stopped in step (c) by lowering the pH to about 11.
Claim 9
Independent Claim
24 words
A method according to any one of the preceding claims, wherein the pH is lowered by adding a. weak acid to the denatured samples.
Claim 10
Dependent Claim
14 words
A method according to claim 8, wherein the weak acid is a water-soluble sugar.
Claim 11
Independent Claim
26 words
A method according to any one of the preceding claims, wherein a reducing agent is used in step (e) as a protective for the fluorescent dye.
Claim 12
Dependent Claim
44 words
A method according to claim 1, as applied to the detection of DNA damage in white blood cells, which comprises dispersing the blood containing the cells to be tested in ammonium chloride solution; incubating the ammonium chloride solution until red cell lysis is complete;
Claim 13
Dependent Claim
98 words
A kit for quantatively comparing the extent of damage in DNA molecules due to chemical or radiation influences, comprising, in separately packaged units,: (i) a lysing reagent which causes partial cell disruption and DNA release; (ii) an alkaline denaturing reagent able to cause separation of the two strands of DNA; (iii) a weak acid able to lower the pH due to (ii) sufficiently to stop DNA denaturation but without causing precipitation; and (iv) a fluorescent dye which interacts and fluoresces with double-strand DNA but not with single-strand DNA or other cell components at the pH due to (iii).
Claim 14
Dependent Claim
98 words
A kit for quantatively comparing the extent of damage in DNA molecules due to chemical or radiation influences, comprising, in separately packaged units,: (i) a lysing reagent which causes partial cell disruption and DNA release; (ii) an alkaline denaturing reagent able to cause separation of the two strands of DNA; (iii) a weak acid able to lower the pH due to (ii) sufficiently to stop DNA denaturation but without causing precipitation; and (iv) a fluorescent dye which interacts and fluoresces with double-strand DNA but not with single-strand DNA or other cell components at the pH due to (iii).
Claim 15
Dependent Claim
15 words
A kit according to claim 12, including means to shear and homogenize the DNA solution.
Claim 16
Dependent Claim
17 words
A kit according to claim 12 or 13, wherein the weak acid (iii) comprises a water-soluble sugar.
Claim 17
Dependent Claim
19 words
A kit according to any one of claims 12 to 14, wherein the fluorescent dye (iv) comprises ethidium bromide.
Claim 18
Dependent Claim
32 words
A kit according to any one of claims 12 to 15, including a neutral buffer solution containing an osmotic stabilizer for, suspending the isolated cells from which DNA is to be released.
Claim 19
Dependent Claim
19 words
A kit according to any one of claims 12 to 16, which additionally includes a buffered ammonium chloride solution.
Claim 20
Dependent Claim
22 words
A kit according to any one of claims 12 to 17, including a reducing agent as a protective for the fluorescent dye.
Claim 21
Dependent Claim
88 words
A kit for quantatively comparing the extent of damage in DNA molecules due to chemical or radiation influences, comprising in separately packaged units: (I) a solution of ammonium chloride buffered to near neutral pH; (II) a solution of meso-inositol, sodium phosphate and MgCl 2 ; (III) a solution comprising urea, NaOH, cyclohexanediaminetetraace- tate and Na dodecylsulfate; (IVa) and (IVb) two alkaline solutions comprising sodium hydroxide of slightly different densities; (V) a solution comprising glucose; and (VI) a fluorescent dye solution at pH about 11 and comprising ethidium bromide.
Claim 22
Dependent Claim
88 words
A kit for quantatively comparing the extent of damage in DNA molecules due to chemical or radiation influences, comprising in separately packaged units: (I) a solution of ammonium chloride buffered to near neutral pH; (II) a solution of meso-inositol, sodium phosphate and MgCl 2 ; (III) a solution comprising urea, NaOH, cyclohexanediaminetetraace- tate and Na dodecylsulfate; (IVa) and (IVb) two alkaline solutions comprising sodium hydroxide of slightly different densities; (V) a solution comprising glucose; and (VI) a fluorescent dye solution at pH about 11 and comprising ethidium bromide.
References Cited (Backward Citations)
US3798131A
Cited
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