Methods for the isolation of nucleic acids and for quantitative DNA extraction and detection for leukocyte evaluation in blood products
First Claim
1. A method for determining the number of cells of interest in a sample, wherein the cells of interest comprise white blood cells comprising DNA, the method comprising:
- (a) applying a sample comprising cellular material to a filter, wherein the cellular material comprises cells of interest;
(b) lysing the cells of interest to form a cell lysate;
(c) immobilizing the released DNA on or within the filter;
(d) removing the remaining cell lysate from the filter and retaining the DNA;
(e) eluting the DNA from the filter;
(f) quantifying the amount of the DNA eluted from the filter; and
(g) determining the number of cells of interest in the sample based on the determined quantity of the DNA eluted.
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Abstract
A method for isolating nucleic acid which comprises:
- (a) applying a sample comprising cells containing nucleic acid to a filter, whereby the cells are retained as a retentate and contaminants are removed;
- (b) lysing the retentate from step (a) while the retentate is retained by the filter to form a cell lysate containing the nucleic acid;
- (c) filtering the cell lysate with the filter to retain the nucleic acid and remove remaining cell lysate;
- (d) optionally washing the nucleic acid retained by the filter; and
- (e) eluting the nucleic acid, wherein the filter composition and dimensions are selected so that the filter is capable of retaining the cells and the nucleic acid.
Additionally, there is provided a substrate for lysing cells and purifying nucleic acid having a matrix and a coating and an integrity maintainer for maintaining the purified nucleic acid. Also provided is a method of purifying nucleic acid by applying a nucleic acid sample to a substrate having an anionic detergent affixed to a matrix, the substrate physically capturing the nucleic acid, bonding the nucleic acid to a substrate and generating a signal when the nucleic acid bonds to the substrate indicating the presence of the nucleic acid. A kit for purifying nucleic acid containing a coated matrix and an integrity maintenance provider for preserving the matrix and purifying nucleic acid is also provided. Further, there is provided a method for quantifying DNA, such as double-stranded or genomic DNA, isolated from cells, such as leukocytes to determine the numbers of leukocytes in a sample of leukoreduced blood.
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Citations
63 Claims
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1. A method for determining the number of cells of interest in a sample, wherein the cells of interest comprise white blood cells comprising DNA, the method comprising:
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(a) applying a sample comprising cellular material to a filter, wherein the cellular material comprises cells of interest;
(b) lysing the cells of interest to form a cell lysate;
(c) immobilizing the released DNA on or within the filter;
(d) removing the remaining cell lysate from the filter and retaining the DNA;
(e) eluting the DNA from the filter;
(f) quantifying the amount of the DNA eluted from the filter; and
(g) determining the number of cells of interest in the sample based on the determined quantity of the DNA eluted.
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2. A method for determining the number of cells of interest in a sample, wherein the cells of interest comprise white blood cells comprising DNA, the method comprising:
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(a) applying a sample comprising cellular material to a filter, wherein the cellular material comprises cells of interest;
(b) lysing the cells of interest to form a cell lysate;
(c) immobilizing the released DNA on or within the filter;
(d) removing the remaining cell lysate from the filter and retaining the DNA;
(e) quantifying the amount of the DNA retained on the filter; and
(f) determining the number of cells of interest in the sample based on the determined quantity of the DNA retained.
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3. A method for estimating the number of white blood cells in a blood sample from a source of interest, wherein the white blood cells comprise DNA, the method comprising:
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(a) isolating the DNA from the blood sample;
(b) determining the amount of the DNA isolated from the blood sample; and
(c) determining the number of white blood cells in the blood sample based on the determined amount of the isolated DNA. - View Dependent Claims (4, 5, 6, 7, 8, 9, 10)
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11. A method for determining the amount of white blood cells in a blood sample, wherein the white blood cells comprise DNA, the method comprising:
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(a) applying a blood sample comprising cells to a filter in the absence of a chaotrope;
(b) retaining the cells with the filter as a cellular retentate and removing contaminants;
(c) lysing the cellular retentate from step (b) to form a cell lysate while retaining the cell lysate in the filter, the cell lysate retentate containing the DNA;
(d) removing the remaining cell lysate from the filter and retaining the DNA;
(e) eluting the DNA;
(f) measuring the amount of the DNA eluted from the filter; and
(g) determining the amount of the DNA measured to estimate the number of white blood cells in the sample based on an amount of approximately 7 pg of nucleic acid contained in each white blood cell. - View Dependent Claims (12)
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13. A method for isolating DNA from a blood sample comprising:
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(a) applying a blood sample comprising cells to a filter in the absence of a chaotrope;
(b) retaining the cells with the filter as a cellular retentate and removing contaminants;
(c) lysing the cellular retentate from step (b) to form a cell lysate while retaining the cell lysate in the filter, the cell lysate retentate containing DNA;
(d) removing the remaining cell lysate from the filter and retaining the DNA;
(c) heating the DNA to an elevated temperature of 40°
C. to 125°
C. while retained by the filter;
(f) eluting the DNA;
(g) quantifying the amount of DNA eluted from the filter; and
(h) determining the number of DNA-containing cells in the sample based on the determined quantity of the DNA eluted. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
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43. A method for estimating the amount of white blood cells in a leukoreduced blood sample, wherein the white blood cells comprise DNA, the method comprising:
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(a) compressing a filter to between 10% and 80% less than its original volume;
(b) applying a leukoreduced blood sample comprising cells to the filter in the absence of a chaotrope;
(c) retaining the cells with the filter as a cellular retentate comprising whole cells, condensed nuclear material, and cell debris;
(d) removing non-cellular contaminants from the retentate;
(e) lysing the cells in the cellular retentate from step (b) while the retentate is entrapped within the filter to form a cell lysate containing the DNA;
(f) retaining the DNA while removing remaining cell lysate;
(g) heating the DNA to an elevated temperature of 40°
C. to 125°
C. while retained by the filter,(h) eluting the DNA;
(i) measuring the amount of genomic DNA eluted from the filter;
(j) repeating steps (g) and (h); and
(k) determining the amount of DNA measured to estimate the number of white blood cells in the sample based on an amount of approximately 7 pg of DNA contained in each white blood cell. - View Dependent Claims (44, 45, 46, 47, 48)
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49. A kit for quantifying the amount of genomic DNA in a blood sample comprising cells containing DNA comprising:
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(a) a filter supported on a support, said filter comprising matrix means for physically retarding movement of DNA therethrough in the absence of a chaotrope;
(b) one or more solutions selected from the group consisting of a red cell lysis solution, a solution for rupturing intact whole cells to leave condensed nuclear material, a lysis solution for lysing nuclear material and an elution solution; and
(c) an indicator capable of providing quantitative measurement of double-stranded or genomic DNA in a concentration as low as 1000 ng/ml. - View Dependent Claims (50, 51)
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52. A method for estimating the amount of white blood cells in a blood sample, wherein the white blood cells comprise DNA, the method comprising:
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(a) applying a blood sample to a substrate consisting of a coating enabling cellular lysis and release of the DNA, wherein the coating comprises a surfactant or detergent;
(b) immobilizing the released DNA to a matrix, the substrate physically capturing the DNA;
(c) bonding the DNA to the substrate;
(d) generating a signal when the DNA bonds to the substrate;
(e) analyzing the amount of DNA captured by quantifying the generated signal;
(f) estimating by calculation the number of white blood cells in the blood sample based on the amount of DNA isolated. - View Dependent Claims (53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63)
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Specification