Differential enzymatic fragmentation
First Claim
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1. A method of detecting the presence of methylation at a locus within a population of nucleic acids, the method comprising (a) dividing the population of nucleic acids into at least two portions, (b) contacting a first portion with a methylation-sensitive restriction enzyme to obtain a population comprising fragmented unmethylated copies of the locus and intact methylated copies of the locus;
- (c) quantifying the intact copies of the locus in the first portion;
(d) contacting a second portion with a methylation-dependent restriction enzyme to obtain a population comprising fragmented methylated copies of the locus and intact unmethylated copies of the locus;
(e) quantifying the intact copies of the locus in the second portion; and
(f) determining the presence of methylation at the locus by comparing the number of intact copies of the locus in the first portion and number of intact copies of the locus in the second portion.
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Abstract
The present invention provides methods for detecting the presence of methylation at a locus within a population of nucleic acids.
49 Citations
111 Claims
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1. A method of detecting the presence of methylation at a locus within a population of nucleic acids, the method comprising
(a) dividing the population of nucleic acids into at least two portions, (b) contacting a first portion with a methylation-sensitive restriction enzyme to obtain a population comprising fragmented unmethylated copies of the locus and intact methylated copies of the locus; -
(c) quantifying the intact copies of the locus in the first portion;
(d) contacting a second portion with a methylation-dependent restriction enzyme to obtain a population comprising fragmented methylated copies of the locus and intact unmethylated copies of the locus;
(e) quantifying the intact copies of the locus in the second portion; and
(f) determining the presence of methylation at the locus by comparing the number of intact copies of the locus in the first portion and number of intact copies of the locus in the second portion. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 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, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111)
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2. The method of claim 1, further comprising
quantifying a third portion of the nucleic acids, thereby amplifying the total intact copies of the locus in the population; - and
comparing the number of total intact copies of the locus to the number of intact copies of the locus in the first portion and/or intact copies of the locus in the second portion.
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3. The method of claim 1, further comprising contacting a third portion of the nucleic acids with a methylation-dependent restriction enzyme and a methylation-sensitive restriction enzyme;
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quantifying copies of the intact locus in the third portion; and
determining the presence of methylation at the locus by comparing the number of the intact copies of the locus in the third portion to the number of intact copies of the locus in the first portion and/or intact unmethylated copies of the locus in the second portion.
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4. The method of claim 1, wherein the second portion is also contacted with the methylation-sensitive restriction enzyme prior to the step of quantifying the intact copies of the locus in the second portion.
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5. The method of claim 1, wherein the first portion is also contacted with the methylation-dependent restriction enzyme prior to the step of quantifying the intact copies of the locus in the first portion.
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6. The method of claim 2, further comprising contacting a fourth portion of the nucleic acids with a methylation-dependent restriction enzyme and a methylation-sensitive restriction enzyme;
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quantifying intact copies of the locus in the fourth portion; and
determining the presence of methylation at the locus by comparing the number of intact copies of the locus in the fourth portion to the number of total intact copies of the locus in the third portion and/or intact copies of the locus in the first portion and/or intact copies of the locus in the second portion.
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7. The method of claim 2, wherein the second portion is also contacted with the methylation-sensitive restriction enzyme prior to the step of quantifying the intact copies of the locus in the second portion.
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8. The method of claim 2, wherein the first portion is also contacted with the methylation-dependent restriction enzyme prior to the step of quantifying the intact copies of the locus in the first portion.
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9. The method of claim 6, further comprising identification of mutations within the locus.
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10. The method of claim 9, wherein a fifth portion of the nucleic acids is contacted with a methylation-insensitive restriction enzyme;
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quantifying intact copies of the locus in the fifth portion to obtain a population of nucleic acids comprising a mutation at the locus; and
determining the presence of a mutation at the locus by comparing the number of the intact copies of the locus in the fifth portion to the number of intact copies of the locus in the fourth portion and/or total intact copies of the locus in the third portion and/or intact copies of the locus in the first portion and/or intact copies of the locus in the second portion.
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11. The method of claim 2, wherein the number of unmethylated copies of the locus is determined by subtracting the number of intact copies of the locus remaining after the first portion is cut with the methylation-sensitive restriction enzyme from the total intact copies of the locus.
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12. The method of claim 2, wherein the number of methylated copies of the locus is determined by subtracting the number of intact copies of the locus remaining after the second portion is cut with the methylation-dependent restriction enzyme from the total intact copies of the locus.
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13. The method of claim 2, wherein the number of hemimethylated and mutant copies of the locus is determined by:
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(a) subtracting the number of intact copies of the locus remaining after the first portion is cut with the methylation-sensitive restriction enzyme from the total intact copies of the locus, thereby determining the number of unmethylated copies of the locus; and
(b) subtracting the number of unmethylated copies of the locus from the number of intact copies of the locus remaining after the second portion is cut with the methylation-dependent restriction enzyme, thereby determining the number of hemimethylated and mutant copies of the locus.
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14. The method of claim 2, wherein the number of hemimethylated and mutant copies of the locus is determined by:
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(a) subtracting the number of intact copies of the locus remaining after the second portion is cut with the methylation-dependent restriction enzyme from the total intact copies of the locus, thereby determining the number of methylated copies of the locus; and
(b) subtracting the number of methylated copies of the locus from the number of intact copies of the locus remaining after the first portion is cut with the methylation-sensitive restriction enzyme, thereby determining the number of hemimethylated and mutant copies of the locus.
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15. The method of claim 6, wherein the number of methylated and unmethylated copies of the locus is determined by subtracting the number of intact copies of the locus remaining after the fourth portion is cut with the methylation-dependent restriction enzyme and the methylation-sensitive restriction enzyme from the total intact copies of the locus, thereby determining the number of methylated and unmethylated copies of the locus.
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16. The method of claim 10, wherein the number of hemimethylated copies of the locus is determined by subtracting the number of intact loci remaining after the fifth portion of the nucleic acids is contacted with a methylation-insensitive restriction enzyme from the number of intact copies of the locus remaining after the fourth portion is cut with the methylation-dependent restriction enzyme and the methylation-sensitive restriction enzyme.
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17. The method of claim 10, wherein the number of methylated copies of the locus is determined by:
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(a) subtracting the number of intact copies of the locus remaining after the fifth portion of the nucleic acids is contacted with a methylation-insensitive restriction enzyme from the number of intact copies of the locus remaining after the fourth portion is cut with the methylation-dependent restriction enzyme and the methylation-sensitive restriction enzyme, thereby determining the number of hemimethylated copies of the locus; and
(b) subtracting the number of hemimethylated copies of the locus and the number of intact copies of the locus remaining after the fifth portion of the nucleic acids is contacted with the methylation-insensitive restriction enzyme from the number of intact copies of the locus remaining after the first portion of nucleic acids is contacted with the methylation-sensitive restriction enzyme, thereby determining the number of methylated copies of the locus.
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18. The method of claim 10, wherein the number of unmethylated copies of the locus is determined by:
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(a) subtracting the number of intact copies of the locus remaining after the fifth portion of the nucleic acids is contacted with a methylation-insensitive restriction enzyme from the number of intact copies of the locus remaining after the fourth portion is cut with the methylation-dependent restriction enzyme and the methylation-sensitive restriction enzyme, thereby determining the number of hemimethylated copies of the locus; and
(b) subtracting the number of hemimethylated copies of the locus and the number of intact copies of the locus remaining after the fifth portion of the nucleic acids is contacted with the methylation-insensitive restriction enzyme from the number of intact copies of the locus remaining after the second portion of nucleic acids is contacted with the methylation-dependent restriction enzyme, thereby determining the number of unmethylated copies of the locus.
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19. The method of claim 1, wherein the quantifying steps comprise quantitative amplification.
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20. The method of claim 2, wherein the quantifying steps comprise quantitative amplification.
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21. The method of claim 3, wherein the quantifying steps comprise quantitative amplification.
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22. The method of claim 4, wherein the quantifying steps comprise quantitative amplification.
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23. The method of claim 5, wherein the quantifying steps comprise quantitative amplification.
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24. The method of claim 6, wherein the quantifying steps comprise quantitative amplification.
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25. The method of claim 7, wherein the quantifying steps comprise quantitative amplification.
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26. The method of claim 8, wherein the quantifying steps comprise quantitative amplification.
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27. The method of claim 10, wherein the quantifying steps comprise quantitative amplification.
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28. The method of claim 19, wherein the nucleic acids are contacted with an agent that modifies unmethylated cytosines before the amplification step;
and the intact copies of the locus are amplified with a pair of oligonucleotide primers comprising at least one primer that distinguishes between modified methylated and unmethylated DNA.
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29. The method of claim 20, wherein the nucleic acids are contacted with an agent that modifies unmethylated cytosines before the amplification step;
and the intact copies of the locus are amplified with a pair of oligonucleotide primers comprising at least one primer that distinguishes between modified methylated and unmethylated DNA.
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30. The method of claim 21, wherein the nucleic acids are contacted with an agent that modifies unmethylated cytosines before the amplification step;
and the intact copies of the locus are amplified with a pair of oligonucleotide primers comprising at least one primer that distinguishes between modified methylated and unmethylated DNA.
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31. The method of claim 22, wherein the nucleic acids are contacted with an agent that modifies unmethylated cytosines before the amplification step;
and the intact copies of the locus are amplified with a pair of oligonucleotide primers comprising at least one primer that distinguishes between modified methylated and unmethylated DNA.
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32. The method of claim 23, wherein the nucleic acids are contacted with an agent that modifies unmethylated cytosines before the amplification step;
and the intact copies of the locus are amplified with a pair of oligonucleotide primers comprising at least one primer that distinguishes between modified methylated and unmethylated DNA.
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33. The method of claim 24, wherein the nucleic acids are contacted with an agent that modifies unmethylated cytosines before the amplification step;
and the intact copies of the locus are amplified with a pair of oligonucleotide primers comprising at least one primer that distinguishes between modified methylated and unmethylated DNA.
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34. The method of claim 27, wherein the nucleic acids are contacted with an agent that modifies unmethylated cytosines before the amplification step;
and the intact copies of the locus are amplified with a pair of oligonucleotide primers comprising at least one primer that distinguishes between modified methylated and unmethylated DNA.
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35. The method of claim 28, wherein the agent is sodium bisulfite.
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36. The method of claim 29, wherein the agent is sodium bisulfite.
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37. The method of claim 30, wherein the agent is sodium bisulfite.
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38. The method of claim 31, wherein the agent is sodium bisulfite.
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39. The method of claim 32, wherein the agent is sodium bisulfite.
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40. The method of claim 33, wherein the agent is sodium bisulfite.
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41. The method of claim 34, wherein the agent is sodium bisulfite.
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42. The method of claim 1, wherein the methylation-dependent restriction enzyme is contacted to the second portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-dependent restriction enzyme in the locus to remain uncleaved.
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43. The method of claim 2, wherein the methylation-dependent restriction enzyme is contacted to the second portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-dependent restriction enzyme in the locus to remain uncleaved.
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44. The method of claim 3, wherein the methylation-dependent restriction enzyme is contacted to the third portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-dependent restriction enzyme in the locus to remain uncleaved.
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45. The method of claim 6, wherein the methylation-dependent restriction enzyme is contacted to the fourth portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-dependent restriction enzyme in the locus to remain uncleaved.
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46. The method of claim 4, wherein the methylation-dependent restriction enzyme is contacted to the second portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-dependent restriction enzyme in the locus to remain uncleaved.
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47. The method of claim 7, wherein the methylation-dependent restriction enzyme is contacted to the second portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-dependent restriction enzyme in the locus to remain uncleaved.
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48. The method of claim 42, wherein the density of methylation at the locus is determined by comparing the number of intact methylated loci in the second portion after cleavage with a control value representing the quantity of methylation in a control DNA.
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49. The method of claim 43, wherein the density of methylation at the locus is determined by comparing the number of intact methylated loci in the second portion after cleavage with a control value representing the quantity of methylation in a control DNA.
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50. The method of claim 44, wherein the density of methylation at the locus is determined by comparing the number of intact methylated loci in the third portion after cleavage with a control value representing the quantity of methylation in a control DNA.
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51. The method of claim 45, wherein the density of methylation at the locus is determined by comparing the number of intact methylated loci in the fourth portion after cleavage with a control value representing the quantity of methylation in a control DNA
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52. The method of claim 46, wherein the density of methylation at the locus is determined by comparing the number of intact methylated loci in the second portion after cleavage with both enzymes to a control value representing the quantity of methylation in a control DNA.
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53. The method of claim 47, wherein the density of methylation at the locus is determined by comparing the number of intact methylated loci in the second portion after cleavage with both enzymes to a control value representing the quantity of methylation in a control DNA.
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54. The method of claim 1, wherein the methylation-sensitive restriction enzyme is contacted to the first portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-sensitive restriction enzyme in the locus to remain uncleaved.
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55. The method of claim 2, wherein the methylation-sensitive restriction enzyme is contacted to the first portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-sensitive restriction enzyme in the locus to remain uncleaved.
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56. The method of claim 3, wherein the methylation-sensitive restriction enzyme is contacted to the third portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-sensitive restriction enzyme in the locus to remain uncleaved.
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57. The method of claim 5, wherein the methylation-sensitive restriction enzyme is contacted to the first portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-sensitive restriction enzyme in the locus to remain uncleaved.
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58. The method of claim 6, wherein the methylation-sensitive restriction enzyme is contacted to the third portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-sensitive restriction enzyme in the locus to remain uncleaved.
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59. The method of claim 8, wherein the methylation-sensitive restriction enzyme is contacted to the third portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-sensitive restriction enzyme in the locus to remain uncleaved.
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60. The method of claim 54, wherein the density of methylation at the locus is determined by comparing the number of intact unmethylated loci in the first portion after cleavage with a control value representing the quantity of methylation in a control DNA.
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61. The method of claim 55, wherein the density of methylation at the locus is determined by comparing the number of intact unmethylated loci in the first portion after cleavage with a control value representing the quantity of methylation in a control DNA.
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62. The method of claim 56, wherein the density of methylation at the locus is determined by comparing the number of intact unmethylated loci in the third portion after cleavage with a control value representing the quantity of methylation in a control DNA.
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63. The method of claim 57, wherein the density of methylation at the locus is determined by comparing the number of intact unmethylated loci in the first portion after cleavage with both enzymes to a control value representing the quantity of methylation in a control DNA.
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64. The method of claim 58, wherein the density of methylation at the locus is determined by comparing the number of intact unmethylated loci in the third portion after cleavage with a control value representing the quantity of methylation in a control DNA.
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65. The method of claim 59, wherein the density of methylation at the locus is determined by comparing the number of intact unmethylated loci in the first portion after cleavage with both enzymes to a control value representing the quantity of methylation in a control DNA.
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66. The method of claim 3, wherein the methylation-dependent restriction enzyme is contacted with the third portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-dependent restriction enzyme in the locus to remain uncleaved.
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67. The method of claim 66, wherein the density of methylation at the locus is determined by comparing the number of intact methylated loci in the fourth portion after cleavage with a control value representing the quantity of methylation in a control DNA.
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68. The method of claim 3, wherein the methylation-sensitive restriction enzyme is contacted with the third portion under conditions that allow for at least some copies of potential restriction enzyme cleavage sites for the methylation-sensitive restriction enzyme in the locus to remain uncleaved.
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69. The method of claim 68, wherein the density of methylation at the locus is determined by comparing the number of intact unmethylated loci in the fourth portion after cleavage with a control value representing the quantity of methylation in a control DNA.
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70. The method of claim 1, wherein the methylation is at the C4 position of a cytosine within the locus.
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71. The method of claim 1, wherein the methylation is at the C5 position of a cytosine within the locus.
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72. The method of claim 1, wherein the methylation is at the N6 position of an adenosine within the locus.
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73. The method of claim 1, wherein the nucleic acids are DNA.
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74. The method of claim 73, wherein the DNA is genomic DNA.
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75. The method of claim 1, wherein the methylation-sensitive restriction enzyme does not cut when a cytosine within the recognition sequence is methylated at position C5.
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76. The method of claim 75, wherein the methylation-sensitive restriction enzyme is selected from the group consisting of:
- Aat II, Aci I, Acl I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I, Cla I, Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinC II, Hpa II, Hpy99 L HpyCH4 IV, Kas I, Mbo I, Mlu I, MapA1 I, Msp I, Nae I, Nar I, Not I, Pml I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I, Sfo I, SgrA I, Sma I, SnaB I, Tsc I, Xma I, and Zra I.
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77. The method of claim 1, wherein the methylation-sensitive restriction enzyme does not cut when an adenosine within the recognition sequence is methylated at position N6.
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78. The method of claim 77, wherein the methylation-sensitive restriction enzyme is Mbo I.
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79. The method of claim 1, wherein the methylation-dependent restriction enzyme is McrBC.
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80. The method of claim 1, wherein the methylation-dependent restriction enzyme is selected from the group consisting of:
- McrA, MrrA, and DpnI.
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81. The method of claim 1, wherein the first portion of genomic nucleic acids is further contacted with at least a second methylation-sensitive enzyme.
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82. The method of claim 1, wherein the second portion of genomic nucleic acids is further contacted with at least a second methylation-dependent enzyme.
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83. The method of claim 1, wherein the methylation-sensitive restriction enzyme is methyl-adenosine sensitive.
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84. The method of claim 1, wherein the methylation-sensitive restriction enzyme is methyl-cytosine sensitive.
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85. The method of claim 1, further comprising contacting a third portion of the nucleic acids with a second methylation-sensitive restriction enzyme and contacting a fourth portion of the nucleic acids with a second methylation-dependent restriction enzyme;
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quantifying intact loci in the third and fourth portions; and
determining the presence of methylation at the locus by comparing the number of intact copies of the locus in the third and fourth portions to the number of total intact copies of the locus and/or intact methylated copies of the locus and/or intact unmethylated copies of the locus.
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86. The method of claim 85, wherein the first methylation-sensitive restriction enzyme is methyl-cytosine sensitive;
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the second methylation-sensitive restriction enzyme is methyl-adenosine sensitive;
the first methylation-dependent restriction enzyme is methyl-cytosine sensitive; and
the second methylation dependent enzyme is methyl-adenosine sensitive.
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87. The method of claim 1, wherein the presence of methylation at the locus is compared between at least two nucleic acid samples.
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88. The method of claim 87, wherein the at least two nucleic acid samples are isolated from at least two organisms having the same phenotype.
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89. The method of claim 87, wherein the at least two nucleic acid samples are isolated from at least two organisms having different phenotypes.
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90. The method of claim 87, comprising quantifying the intact methylated copies of the locus in a first sample and a second sample;
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comparing the quantity of amplified products from the two samples, thereby determining relative methylation at the locus between the two samples.
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91. The method of claim 87, wherein a first nucleic acid sample is isolated from a cell suspected of being a cancer cell and a second nucleic acid sample is isolated from a non-cancerous cell.
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92. The method of claim 1, wherein the presence of methylation at two or more loci is determined by
quantifying copies of intact DNA at the two different loci from a first portion contacted with a methylation-dependent restriction enzyme; -
quantifying the intact DNA as the two loci from a second portion contacted with a methylation-sensitive restriction enzyme; and
comparing the quantities of the amplified products for the two loci.
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93. The method of claim 1, wherein the population of nucleic acids is isolated from a cell.
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94. The method of claim 93, wherein the cell is a member selected from the group consisting of:
- a plant cell, a fungal cell, a prokaryotic cell, an animal cell, a mammalian cell, and a cancer cell.
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95. The method of claim 1, wherein the population of nucleic acids is isolated from a sample from a subject, wherein the sample is a member selected from the group consisting of:
- a body fluid and a secretion.
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96. The method of claim 95, wherein the subject is a human.
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97. The method of claim 96, wherein the subject is suspected of having cancer.
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98. The method of claim 1, wherein the population of nucleic acids is isolated from a tissue biopsy from a subject.
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99. The method of claim 98, wherein the subject is a human.
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100. The method of claim 98, wherein the subject is suspected of having cancer.
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101. The method of claim 98, wherein the locus comprises a sequence that is more methylated in diseased cells than in non-diseased cells.
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102. The method of claim 98, wherein the locus comprises a sequence that is less methylated in diseased cells than in non-diseased cells.
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103. The method of claim 1, wherein the quantifying step comprises a quantitative polymerase chain reaction (PCR).
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104. The method of claim 103, wherein the quantitative amplification product is detected by detection of a label intercalated between bases of double stranded DNA sequences.
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105. The method of claim 103, wherein the quantitative amplification product is detected by detecting hybridization of a detectably labeled oligonucleotide to the amplification product.
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106. The method of claim 105, wherein the detectably labeled oligonucleotide is a labeled oligonucleotide probe comprising a fluorophore and a hairpin structure.
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107. The method of claim 105, wherein the detectably labeled oligonucleotide is a dual labeled oligonucleotide probe comprising a pair of interactive labels.
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108. The method of claim 107, wherein the pair of interactive labels are a quencher and a fluorophore.
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109. The method of claim 107, wherein the fluorophore is activated to generate a detectable signal when the probe hybridizes to its target nucleic acid sequence.
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110. The method of claim 107, wherein the fluorophore is activated to generate a detectable signal when the probe hybridizes to its target nucleic acid sequence and an enzyme with 5′
- exonuclease activity cleaves the portion of the probe comprising the quencher.
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111. The method of claim 1, further comprising adding sequence tags onto the ends of a population of nucleic acids before dividing the population of nucleic acids into equal portions, wherein,
step (c) comprises quantifying the remaining intact methylated copies of the locus in the first portion with primers that initiate amplification from the sequence tags; - and
step (e) comprises quantifying the remaining unmethylated intact copies of the locus in the second portion with primers that initiate amplification from the sequence tags.
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2. The method of claim 1, further comprising
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Specification
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Current AssigneeOrion Genomics LLC
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Original AssigneeOrion Genomics LLC
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InventorsLakey, Nathan D., Jeddeloh, Jeffrey A.
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Granted Patent
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Time in Patent OfficeDays
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Field of Search
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US Class Current435/6
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CPC Class CodesC12Q 1/683 involving restriction enzym...C12Q 1/6858 Allele-specific amplificationC12Q 2521/331 Methylation site specific n...C12Q 2545/101 with an internal standard/c...