IPC분류정보
국가/구분 |
United States(US) Patent
등록
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국제특허분류(IPC7판) |
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출원번호 |
US-0908452
(2001-07-18)
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우선권정보 |
GB-0018193 (1999-08-02); GB-0022141 (1998-10-09) |
발명자
/ 주소 |
- Fomovskaia, Galina N.
- Smith, Martin A.
- Fomovsky, Mikhail A.
- Butt, Neil J.
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출원인 / 주소 |
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대리인 / 주소 |
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인용정보 |
피인용 횟수 :
76 인용 특허 :
12 |
초록
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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 ce
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.
대표청구항
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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:(a) applying a sample comprising cellular material to a filter, wherein the cellular material comprises cells of interest; (b) lysin
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. 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:(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. 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:(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. 4. The method according to claim 3, wherein the blood sample is a leukoreduced blood sample.5. The method according to claim 4, wherein the DNA is genomic DNA, double-stranded DNA, or single-stranded DNA.6. The method according to claim 4, wherein the estimated number of white blood cells is at least 85% of the number of white blood cells in a second blood sample as measured by flow cytometry, the method further comprising:(d) obtaining a second blood sample from the source of interest; and (e) measuring the number of white blood cells in the second blood sample using flow cytometry; and (f) comparing the number of white blood cells estimated for the blood sample in step (c) with the number measured in the second blood sample in step (e). 7. The method according to claim 6, wherein the estimated number of white blood cells is at least 90% of the number of white blood cells as measured by flow cytometry.8. The method according to claim 6, wherein the estimated number of white blood cells is at least 95% of the number of white blood cells as measured by flow cytometry.9. The method according to claim 6, wherein the estimated number of white blood cells is at least 98% of the number of white blood cells as measured by flow cytometry.10. The method according to claim 6, wherein the estimated number of white blood cells is at least 99% of the number of white blood cells as measured by flow cytometry.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:(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. 12. The method according to claim 11, wherein the DNA is genomic DNA, double-stranded DNA, or single-stranded DNA.13. A method for isolating DNA from a blood sample comprising:(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. 14. The method according to claim 13, wherein steps (e) and (f) together are repeated at least once.15. The method according to claim 13, wherein the elevated temperature is in the range of 80° C. to 95° C.16. The method according to claim 13, further comprising the method wherein, prior to step (a), the filter is compressed to between 10% and 80% less than its original volume.17. The method according to claim 16, wherein the filter is compressed to between 25% and 75% less than its original volume.18. The method according to claim 16, wherein the filter is compressed to between 40% and 60% less than its original volume.19. The method according to claim 13, wherein the cellular retentate comprises intact whole blood cells.20. The method according to claim 13, wherein the cell lysate retentate comprises condensed material from the nucleus and blood cell debris.21. The method according to claim 13, wherein step (c) further comprises lysing the cellular retentate to form a cell lysate containing the nucleic acid by the addition of a low salt buffer capable of lysing the cells, followed by the removal of the low salt buffer.22. The method according to claim 21, wherein step (b) comprises:(a) rupturing the intact whole cells retained by the filter to leave condensed material from the nucleus retained by the filter; and (b) lysing the condensed material from the nucleus to form the cell lysate containing the DNA. 23. The method according to claim 22, wherein the intact whole cells are ruptured to form condensed material from the nucleus by addition of detergent.24. The method according to claim 13, wherein the filter composition and dimensions are selected so that the DNA is retained by the filter in step (c) by non-ionic interaction.25. The method according to claim 13, wherein the retaining step further comprises physically retarding the movement of the DNA through the filter.26. The method according to claim 13, wherein the filter composition and dimensions are selected so that the DNA is retained by the filter in step (c) in the form of a web.27. The method according to claim 13, wherein the filter comprises a plurality of fibers and has a disordered structure.28. The method according to claim 27, wherein the fiber diameters are in the range of 1 μm to 10 μm.29. The method according to claim 13, wherein the cells are white blood cells.30. The method according to claim 29, which method further comprises applying blood to the solid phase, lysing the red blood cells therefrom, washing the solid phase to remove contaminants and obtaining the cell lysate from the white blood cells.31. The method according to claim 30, wherein the blood is leukoreduced blood.32. The method according to claim 31, wherein the estimated number of white blood cells is at least 85% of the number of white blood cells in a second blood sample as measured by flow cytometry, the method further comprising:(i) obtaining a second blood sample from the source of interest; and (j) measuring the number of white blood cells in the second blood sample using flow cytometry; and (k) comparing the number of white blood cells determined for the blood sample in step (h) with the number measured in the second blood sample in step (j). 33. The method according to claim 32, wherein the estimated number of white blood cells is at least 90% of the number of white blood cells as measured by flow cytometry.34. The method according to claim 32, wherein the estimated number of white blood cells is at least 95% of the number of white blood cells as measured by flow cytometry.35. The method according to claim 32, wherein the estimated number of white blood cells is at least 98% of the number of white blood cells as measured by flow cytometry.36. The method according to claim 32, wherein the estimated number of white blood cells is at least 99% of the number of white blood cells as measured by flow cytometry.37. The method according to claim 13, wherein the DNA is genomic DNA, double-stranded DNA, or single-stranded DNA.38. The method according to claim 13, which is carried out without any centrifugation steps.39. The method according to claim 13, which is carried out in the absence of a chaotrope.40. The method according to claim 13, wherein step (g) further comprises detecting the presence of the DNA using a fluorescent signal, color indicator, photometric indicator, enzymatic indicator, or radioactive indicator.41. The method according to claim 13, wherein step (g) further comprises detecting the presence of the DNA using an indicator specific for double-stranded DNA.42. The method according to claim 13, wherein step (g) further comprises detecting the presence of the DNA using an indicator from the group consisting of PicoGreen, a labeled nucleic acid probe specific for genomic DNA, ethidium bromide, or an indicator specific for double-stranded DNA.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:(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. 44. The method according to claim 43, wherein the estimated number of white blood cells is at least 85% of the number of white blood cells in a second leukoreduced blood sample as measured by flow cytometry, the method further comprising:(l) obtaining a second blood sample from the source of interest; and (m) measuring the number of white blood cells in the second blood sample using flow cytometry; and (n) comparing the number of white blood cells estimated for the blood sample in step (k) with the number measured in the second blood sample in step (m). 45. The method according to claim 44, wherein the estimated number of white blood cells is at least 90% of the number of white blood cells as measured by flow cytometry.46. The method according to claim 44, wherein the estimated number of white blood cells is at least 95% of the number of white blood cells as measured by flow cytometry.47. The method according to claim 44, wherein the estimated number of white blood cells is at least 98% of the number of white blood cells as measured by flow cytometry.48. The method according to claim 44, wherein the estimated number of white blood cells is at least 99% of the number of white blood cells as measured by flow cytometry.49. A kit for quantifying the amount of genomic DNA in a blood sample comprising cells containing DNA comprising:(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. 50. The kit according to claim 49, wherein the indicator is capable of providing quantitative measurement of double-stranded or genomic DNA in a concentration as low as 200 pg/ml.51. The kit according to claim 49, wherein the blood sample is a leukoreduced blood sample.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:(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. 53. The method according to claim 54, wherein the generating step further comprises generating a fluorescent signal, color indicator, photometric indicator, enzymatic indicator, or radioactive indicator.54. The method according to claim 52, wherein the blood sample is a leukoreduced blood sample.55. The method according to claim 54, wherein the estimated number of white blood cells is at least 85% of the number of white blood cells in a second sample as measured by flow cytometry, the method further comprising:(g) obtaining a second blood sample from the source of interest; and (h) measuring the number of white blood cells in the second blood sample using flow cytometry; and (i) comparing the number of white blood cells estimated for the blood sample in step (f) with the number measured in the second blood sample in step (h). 56. The method according to claim 55, wherein the estimated number of white blood cells is at least 90% of the number of white blood cells as measured by flow cytometry.57. The method according to claim 55, wherein the estimated number of white blood cells is at least 95% of the number of white blood cells as measured by flow cytometry.58. The method according to claim 55, wherein the estimated number of white blood cells is at least 98% of the number of white blood cells as measured by now cytometry.59. The method according to claim 55, wherein the estimated number of white blood cells is at least 99% of the number of white blood cells as measured by flow cytometry.60. The method according to claim 52, wherein the coating further comprises:(i) a weak base; and (ii) a chelating agent. 61. The method according to claim 60, wherein the coating further comprises:(iii) uric acid or a urate salt. 62. The method according to claim 52, wherein the surfactant or detergent comprises sodium dodecyl sulfate.63. The method according to claim 60, wherein:(i) the weak base comprises Tris; and (ii) the chelating agent comprises ethylenediaminetetra-acetic acid.
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