METHOD FOR PROVIDING DNA FRAGMENTS DERIVED FROM A REMOTE SAMPLE
First Claim
Patent Images
1. A method for providing DNA fragments derived from a remote sample, comprising:
- providing a remote sample comprising DNA;
isolating DNA from the remote sample; and
treating the isolated DNA with a reagent or enzyme which allows differentiation of methylated and unmethylated cytosine.
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Accused Products
Abstract
Aspects of the present invention relate to compositions and methods for providing DNA fragments from a remote sample. In particular aspects a remote sample comprising DNA is provided, DNA is isolated from the remote sample, and the isolated DNA is treated in a way which allows differentiation of methylated and unmethylated cytosine. Additional, particular embodiments provide compositions and methods for methylation analysis of DNA derived from a remote sample. Other aspects provide for compositions and methods of whole genome amplification of bisulfite treated DNA.
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Citations
71 Claims
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1. A method for providing DNA fragments derived from a remote sample, comprising:
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providing a remote sample comprising DNA; isolating DNA from the remote sample; and treating the isolated DNA with a reagent or enzyme which allows differentiation of methylated and unmethylated cytosine. - 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, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58, 61, 62, 63, 64)
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2. The method of claim 1, wherein the DNA of the remote sample is characterized in that less than about 5%, less than about 3%, less than about 1%, or less than about 0.1% of the DNA is derived from a defined cell, group of cells, tissue or organ.
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3. The method of claim 1, wherein the remote sample is characterized in that it comprises less than about 100 ng DNA in 1 ml, less than about 60 ng DNA in 1 ml or less than about 10 ng DNA in 1 ml.
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4. The method of claim 1, wherein loss of DNA is minimized by at least one method selected from the group consisting of:
- selection of a DNA isolation method characterized by high yields of DNA;
selection of a method for differentiation of unmethylated and methylated cytosine characterized by high accuracy and high reliability;
high accuracy of pipetting;
reuse of pipetting device; and
reuse of device contacted with DNA.
- selection of a DNA isolation method characterized by high yields of DNA;
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5. The method of claim 1, wherein the volume of the remote sample is at least about 1.5 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 11 ml, about 12 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 40 ml, or about 50 ml.
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6. The method of claim 1, wherein the remote sample is at least one sample selected from the group comprising:
- blood sample, plasma sample, serum sample, body fluid sample, saliva sample, urine sample, semen sample, sample of the fluid from the pleural cavity, sample from the fluid from the peritoneal cavity, sample of the cerebrospinal fluid, smear from a epithelial surface, sputum sample, stool sample, ejaculate sample, tears sample, sweat sample, lymph fluid sample, bronchial lavage sample, pleural effusion sample, meningal fluid sample, glandular fluid sample, fine needle aspirates sample, nipple aspirates fluid sample, spinal fluid sample, conjunctival fluid sample, vaginal fluid sample, duodenal fluid sample, prancreatic juice sample, and bile sample.
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7. The method of claim 1, wherein the remote sample is plasma, and the providing of the remote sample comprises one or more of the following:
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obtaining at least about 5 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml of blood from a individual; adding EDTA (ethylene-diamine-tetra-acetic acid) to the blood comprising gentle mixing; adjusting the blood to a final concentration of about 2.2 μ
mol/l, about 3.2 μ
mol/l, about 3.7 μ
mol/l, about 4.0 μ
mol/l, about 4.5 μ
mol/l, about 4.9 μ
mol/l, about 5.4 μ
mol/l, about 5.9 μ
mol/l, or about 6.9 μ
mol/l, dipotassium EDTA (dipotassium ethylene-diamine-tetra-acetic acid) comprising gently mixing;centrifuging the blood-EDTA mixture at about 750×
g, about 1000×
g, about 1500×
g, or about 2000×
g for about 4 min, about 8 min, about 10 min, about 12 min, or about 20 min at about 1°
C., about 4°
C., about 7°
C., about 10°
C., about 15°
C., about 21°
C., or about 27°
C.;transferring the plasma into a new container; centrifuging the plasma at about 750×
g, about 1000×
g, about 1500×
g, or about 2000×
g for about 4 min, about 8 min, about 10 min, about 12 min, or about 20 min at about 1°
C., about 4°
C., about 7°
C., or about 10°
C.;transferring the re-centrifuged plasma into a new container; cooling a plasma comprising sample at about 0°
C., about 2°
C., about 4°
C., about 6°
C., or about 10°
C.;freezing, storing or transporting a plasma comprising sample at least at about −
10°
C., about −
20°
C., about −
50°
C., about −
60°
C., about −
70°
C., about −
80°
C., about −
90°
C., or about −
196°
C.; andperforming the providing of the remote sample from obtaining blood from a individual to freezing the corresponding re-centrifuged plasma within about 1, about 2, about 3, about 4, about 5, about 6, or about 8 hours.
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8. The method of claim 1, wherein the remote sample is urine and the providing of the remote sample comprises one or more of the following:
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performing prostatic palpation, prostatic massage, or both from the middle of the prostate to the left side of the prostate, to the right side of the prostate or both for about 10 s, about 30 s, about 50 s, about 60 s, about 75 s, or about 120 s; collecting the first about 5 ml, about 10 ml about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 40 ml of voided urine; adding EDTA to the urine; adjusting the urine to a final concentration of about 3 mmol/l, about 6 mmol/l, about 7 mmol/l, about 8 mmol/l, about 9 mmol/l, about 9.80 mmol/l, about 10 mmol/l, about 11 mmol/l, about 12 mmol/l, about 13 mmol/l, about 14 mmol/l, about 18 mmol/l, or about 25 mmol/l EDTA (ethylene-diamine-tetra-acetic acid) with a pH of about 5.0, about 6.0, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 10; cooling the urine comprising sample at about 0°
C., about 2°
C., about 4°
C., about 6°
C., or about 10°
C.;freezing, storing or transporting the urine comprising sample at least at about −
20°
C., about −
50°
C., about −
60°
C., about −
70°
C., about −
80°
C., about −
90°
C., or about −
196°
C.; andperforming the providing of the urine sample from collecting the first ml of voided urine to freezing the corresponding urine-EDTA mixture within about 15, about 30, about 45, about 60, about 75, about 90, or about 120 min.
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9. The method of claim 1, wherein the remote sample is divided into different subsamples subsequent to providing the remote sample.
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10. The method of claim 1, wherein the remote sample or at least one component of the remote sample is concentrated subsequent to providing the remote sample.
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11. The method of claim 10, wherein the concentration comprises ultrafiltration, volume reduction, or both.
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12. The method of claim 1, wherein the isolation of DNA comprises one or more of the following:
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treating the remote sample with a protease; treating the remote sample with at least one protein degenerating reagent or solution; bringing the DNA of the remote sample in contact with a DNA-purifying device; washing the DNA on the DNA-purifying device; and recovering the DNA from the DNA-purifying device.
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13. The method of claim 12, wherein the protease is at least one selected from the group consisting of:
- serine protease, thiol protease, carboxy protease, metalloprotease, and proteinase K.
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14. The method of claim 12, wherein the DNA purifying device is at least one selected from the group consisting of:
- ultrafiltration, Microcon filter device, filter device, silica surface, silica membrane, magnetic particle, polystyrol particle, polystyrol surface, positively charged surface, and positively charged membrane, charged membrane, charged surface, charged switch membrane, charged switched surface, column of the ZR DNA Clean &
Concentrator-5 Kit, column of the Wizard Genomic DNA Purification Kit, column of the QIAamp DNA Micro Kit, a component of the MagNA Pure Compact Nucleic Acid Isolation Kit (I) Large Volume, a component of the QIAamp UltraSens Virus Kit, a component of the RTP DNA/RNA Virus Supersense Kit, a component of the chemagic Viral DNA/RNA Kit special, a component of the chemagic DNA Blood Kit special, a component of the High Pure Viral Nucleic Acid Kit, a component of the Puregene DNA Isolation Kit, a component of the MasterPure™
Complete DNA and RNA Purification Kit, a component of the NucliSens®
Isolation Kit, and equivalents thereof.
- ultrafiltration, Microcon filter device, filter device, silica surface, silica membrane, magnetic particle, polystyrol particle, polystyrol surface, positively charged surface, and positively charged membrane, charged membrane, charged surface, charged switch membrane, charged switched surface, column of the ZR DNA Clean &
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15. The method of claim 12, wherein the isolation of DNA is carried out by use of at least one kit selected from the group consisting of:
- MagNA Pure Compact Nucleic Acid Isolation Kit (I) Large Volume, QIAamp UltraSens Virus Kit, and RTP DNA/RNA Virus Supersense Kit, chemagic Viral DNA/RNA Kit special, chemagic DNA Blood Kit special, High Pure Viral Nucleic Acid Kit, Puregene DNA Isolation Kit, MasterPure™
Complete DNA and RNA Purification Kit, or NucliSens®
Isolation Kit.
- MagNA Pure Compact Nucleic Acid Isolation Kit (I) Large Volume, QIAamp UltraSens Virus Kit, and RTP DNA/RNA Virus Supersense Kit, chemagic Viral DNA/RNA Kit special, chemagic DNA Blood Kit special, High Pure Viral Nucleic Acid Kit, Puregene DNA Isolation Kit, MasterPure™
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16. The method of claim 1, wherein isolated DNA derived from different samples is pooled, concentrated or pooled and concentrated.
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17. The method of claim 16, wherein the concentration of isolated DNA comprises at least one method elected from the group consisting of:
- ultrafiltration, Microcon filter device, filter device, ethanol precipitation, propanol precipitation, silica surface, silica membrane, magnetic particle, polystyrol particle, positively charged surface, and positively charged membrane, charged membrane, charged surface, charged switch membrane, charged switched surface, vacuum concentration, vacuum concentration by means of a centrifuge, column of the ZR DNA Clean &
Concentrator-5 Kit, column of the Wizard Genomic DNA Purification Kit, column of the QIAamp DNA Micro Kit, a component of the MagNA Pure Compact Nucleic Acid Isolation Kit (I) Large Volume, a component of the QIAamp UltraSens Virus Kit, a component of the RTP DNA/RNA Virus Supersense Kit, a component of the chemagic Viral DNA/RNA Kit special, a component of the chemagic DNA Blood Kit special, a component of the High Pure Viral Nucleic Acid Kit, a component of the Puregene DNA Isolation Kit, a component of the MasterPure™
Complete DNA and RNA Purification Kit, a component of the NucliSens®
Isolation Kit, and equivalents thereof.
- ultrafiltration, Microcon filter device, filter device, ethanol precipitation, propanol precipitation, silica surface, silica membrane, magnetic particle, polystyrol particle, positively charged surface, and positively charged membrane, charged membrane, charged surface, charged switch membrane, charged switched surface, vacuum concentration, vacuum concentration by means of a centrifuge, column of the ZR DNA Clean &
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18. The method of claim 1, wherein the reagent which allows differentiation of methylated and unmethylated cytosine is a reagent that converts unmethylated cytosine to uracil and leaves methylated cytosine unchanged.
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19. The method of claim 1, wherein the reagent that converts unmethylated cytosine to uracil and leaves methylated cytosine unchanged is a bisulfite reagent.
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20. The method of claim 19, wherein treating DNA with a bisulfite reagent comprises:
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mixing of about 10 to about 250 μ
l of a solution comprising DNA with about 45 to about 750 μ
l of bisulfite solution, the bisulfite solution having a pH in the range of about 5.45 to about 5.50 comprising about 4.83 to about 4.93 mol/l hydrogensulfite;adding about 5 to about 500 μ
l of an organic radical scavenger solution, the organic radical scavenger solution comprising an organic solvent and about 10 to about 750 mmol/l of 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid; andapplying a temperature protocol for about 2 to about 18 h, wherein the reaction is conducted in a temperature range of about 0 to about 80°
C. with about 2 to about 5 additional temperature increases, in each case for about 0.5 to about 10 min, to a temperature of about 85 to about 100°
C. including an initial temperature increase to a temperature of about 85 to about 100°
C.
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21. The method of claim 20, comprising:
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mixing about 50 to about 150 μ
l of solution comprising DNA with about 177 to about 531 μ
l of the bisulfite solution;adding about 73 to about 219 μ
l of dioxane solution, the dioxane solution comprising about 157 mmol/l of 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid dissolved in 1,4-dioxane; andapplying a temperature protocol for about 3 to about 16 h, wherein the reaction is conducted in a temperature range of about 57 to about 65°
C. with about 2 to about 5 additional temperature increases, in each case for about 3 to about 5 min, to a temperature of about 94 to about 100°
C. including an initial temperature increase to a temperature of about 94 to about 100°
C.
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22. The method of claim 20, comprising:
-
mixing of about 50 to about 150 μ
l of a solution containing the DNA with about 95 to about 285 μ
l of the bisulfite solution;adding about 15 to about 45 μ
l of DME solution, the DME solution comprising about 500 mmol/l of 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid dissolved in diethyleneglycoldimethylether; andapplying a temperature protocol for about 3 to about 16 h, wherein the reaction is conducted in a temperature range of about 57 to about 65°
C. with about 2 to about 5 additional temperature increases, in each case for about 3 to about 5 min, to a temperature of about 94 to about 100°
C. including an initial temperature increase to a temperature of about 94 to about 100°
C.
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23. The method of claim 1, wherein treating DNA with a reagent or enzyme allowing differentiation of the methylation status comprises purifying the treated DNA.
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24. The method of claim 23, wherein purifying the treated DNA comprises the use of at least one method selected from the group consisting of:
- ultrafiltration, Microcon filter device, filter device, ethanol, propanol, silica surface, silica membrane, magnetic particle, polystyrol particle, positively charged surface, and positively charged membrane, charged membrane, charged surface, charged switch membrane, charged switched surface, column of the ZR DNA Clean &
Concentrator-5 Kit, column of the Wizard Genomic DNA Purification Kit, column of the QIAamp DNA Micro Kit, a component of the MagNA Pure Compact Nucleic Acid Isolation Kit (I) Large Volume, a component of the QIAamp UltraSens Virus Kit, a component of the RTP DNA/RNA Virus Supersense Kit, a component of the chemagic Viral DNA/RNA Kit special, a component of the chemagic DNA Blood Kit special, a component of the High Pure Viral Nucleic Acid Kit, a component of the Puregene DNA Isolation Kit, a component of the MasterPure™
Complete DNA and RNA Purification Kit, or a component of the NucliSens®
Isolation Kit.
- ultrafiltration, Microcon filter device, filter device, ethanol, propanol, silica surface, silica membrane, magnetic particle, polystyrol particle, positively charged surface, and positively charged membrane, charged membrane, charged surface, charged switch membrane, charged switched surface, column of the ZR DNA Clean &
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25. The method of claim 14, comprising:
-
adding of about 50 to about 1000 μ
l of water to the sample after the bisulfite reaction;applying the mixture onto a Microcon filter device subsequently centrifuging at about 10,000 to about 18,000×
g for about 10 to about 30 min;washing with about 100 to about 800 μ
l of about 0.2 mol/l sodium hydroxide, and subsequent centrifuging at about 10,000 to about 18,000×
g for about 6 to about 25 min;applying of about 100 to about 800 μ
l of about 0.1 mol/l sodium hydroxide, and subsequent centrifuging at about 10,000 to about 18,000×
g for about 6 to about 25 min;applying, in 1 to about 8 repetitions, the following;
applying of about 100 to about 400 μ
l water or TE buffer and subsequent centrifuging at about 10,000 to about 18,000×
g for about 6 to about 25 min; andeluting by application of about 25 to about 200 μ
l TE buffer preheated to about 15 to about 65°
C., incubation for about 1 to about 30 min at a temperature of about 15 to about 65°
C., and subsequent inversion of the Microcon filter device and centrifugation at about 500 to about 5,000×
g for about 0.5 to about 30 min.
-
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26. The method of claim 25, comprising:
-
a) adding of 200 μ
l water to the sample after the bisulfite reaction,b) applying the mixture onto a Microcon filter device subsequently centrifuging at about 14,000×
g for about 20 min,c) washing with about 400 μ
l of about 0.2 mol/l sodium hydroxide, and subsequent centrifuging at about 14,000×
g for about 10 to about 14 min,d) applying of about 400 μ
l of about 0.1 mol/l sodium hydroxide, and subsequent centrifuging at about 14,000×
g for about 10 to about 14 min,e) applying, in 1 to about 4 repetitions, the following;
applying of about 400 μ
l water or TE buffer and subsequent centrifuging at about 14,000×
g for about 12 min; andf) eluting by application of about 45 to about 70 μ
l TE buffer preheated to about 50°
C., incubation for about 10 min at a temperature of about 50°
C., and subsequent inversion of the Microcon filter device and centrifugation at about 1,000×
g for about 7 min.
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27. The method of claim 26, further comprising at least one of the following:
-
in step b, applying the mixture in portions onto the Microcon filter device in step b; subsequent to step b, applying of about 400 μ
l TE buffer, the TE buffer pH 8 containing about 10 mmol/l tris-hydroxymethyl-amino-methan and about 0.1 mmol/l EDTA, subsequent centrifuging at about 14,000×
g for about 12 min;in step c, incubating the about 0.2 mol/l sodium hydroxide for about 10 min at room temperature; and in step d, incubating the about 0.1 mol/l sodium hydroxide for about 10 min at room temperature.
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45. The method of claim 1 for determining the methylation status of at least one cytosine, a methylation pattern, or both in the DNA of the remote sample, comprising at least one of the following:
-
determining the methylation status of at least one cytosine in the DNA of the remote sample, each cytosine located at a defined position; and determining a methylation pattern in the DNA of the remote sample.
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46. The method of claim 45, wherein determining of a methylation status, a methylation pattern, or both comprises the use of at least one method selected from the group consisting of:
- amplification method, PCR method, isothermal amplification method, NASBA method, LCR method, methylation specific amplification method, MSP (Methylation Specific PCR) method, nested MSP method, HeavyMethyl™
method, detection method, methylation specific detection method, bisulfite sequencing method, detection by means of DNA-arrays, detection by means of oligonucleotide microarrays, detection by means of CpG-island-microarrays, detection by means of restriction enzymes, simultaneous methylation specific amplification and detection method, COBRA method, real-time PCR, HeavyMethyl™
real time PCR method, MSP MethyLight™
method, MethyLight™
method, MethyLight™
Algo™
method, QM method, Headloop MethyLight™
method, HeavyMethyl™
MethyLight™
method, HeavyMethyl™
Scorpion™
method, MSP Scorpion™
method, Headloop Scorpion™
method, methylation sensitive primer extension, and Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) method.
- amplification method, PCR method, isothermal amplification method, NASBA method, LCR method, methylation specific amplification method, MSP (Methylation Specific PCR) method, nested MSP method, HeavyMethyl™
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47. The method of claim 45 for identification of a marker, further comprising:
-
identification of at least one methylation pattern, comprising the methylation status of at least two CpG positions, wherein said CpG positions are comprised within one DNA fragment and are localized in cis, and wherein the methylation pattern differs between DNA derived from a cell, group of cells, tissue, organ or individual characterized by a condition A and DNA derived from a cell, group of cells, tissue, organ or individual characterized by a condition B; and selecting a cut off value for the percentage of DNA fragments characterized by a identified methylation pattern within a mixture of DNA fragments, wherein a percentage value equal to or larger than the cut off value is indicative for condition A and a percentage value smaller than the cut off value is indicative for condition B, or wherein a percentage value smaller than the cut off value is indicative for condition A and a percentage value equal to or larger than the cut off value is indicative for condition B.
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48. The method of claim 47, wherein the identification of a marker is enabled with at least one of the following:
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a sensitivity of more than about 20%, about 30%, about 35%, about 40% %, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%; and a specificity of more than about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%.
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49. The method of claim 48, wherein a colon cancer marker is identified characterized by at least one of the following:
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a sensitivity of at least about 25%, about 35%, about 40%, about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%; and a specificity of at least about 65%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.
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50. The method of claim 47, wherein the cut off value is selected according to at least one of the following criteria:
-
a sensitivity of more than about 15%, about 25%, about 35%, about 40%, about 50%, about 55%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95%; and a specificity of more than about 20%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75,%, about 80%, about 85%, about 90%, about 95% or about 99%.
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51. The method of claim 47, for at least one of the following:
- diagnosing a condition, providing a prognosis of a condition, predicting treatment response of a condition, determining a predisposition for a condition, predicting a predisposition for a condition, determining a progression of a condition, predicting a progression of a condition, grading a condition, staging a condition, classification of a condition, characterization of a condition, or combinations thereof, wherein the condition is a healthy condition or an adverse event, wherein one of the aforesaid is deduced from the percentage value for DNA fragments characterized by pre-identified methylation pattern within a mixture of DNA fragments, and wherein the corresponding methylation status of CpG positions are measured according to an embodiment described herein, further comprising at least one of the following;
deducing one of the aforesaid for a condition A in case the percentage value is equal to or larger than the selected cut off value; deducing one of the aforesaid for condition B in case the percentage value is smaller than the selected cut off value; deducing one of the aforesaid for condition B in case the percentage value is larger than the selected cut off value; and deducing one of the aforesaid for condition A in case the percentage value is equal to or smaller than the selected cut off value.
- diagnosing a condition, providing a prognosis of a condition, predicting treatment response of a condition, determining a predisposition for a condition, predicting a predisposition for a condition, determining a progression of a condition, predicting a progression of a condition, grading a condition, staging a condition, classification of a condition, characterization of a condition, or combinations thereof, wherein the condition is a healthy condition or an adverse event, wherein one of the aforesaid is deduced from the percentage value for DNA fragments characterized by pre-identified methylation pattern within a mixture of DNA fragments, and wherein the corresponding methylation status of CpG positions are measured according to an embodiment described herein, further comprising at least one of the following;
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52. The method of claim 47, wherein condition A, condition B, or both are a healthy condition or at least one adverse event, the adverse event comprising at least one category selected from the group consisting of:
- undesired drug interactions;
cancer diseases, proliferative diseases or therewith associated diseases;
CNS malfunctions;
damage or disease;
symptoms of aggression or behavioral disturbances;
clinical;
psychological and social consequences of brain damages;
psychotic disturbances and personality disorders;
dementia and/or associated syndromes;
cardiovascular disease of the gastrointestinal tract;
malfunction, damage or disease of the respiratory system;
lesion, inflammation, infection, immunity and/or convalescence;
malfunction, damage or disease of the body as an abnormality in the development process;
malfunction, damage or disease of the skin, of the muscles, of the connective tissue or of the bones;
endocrine and metabolic malfunction, damage or disease; and
headaches or sexual malfunction.
- undesired drug interactions;
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53. A kit for carrying out a method of claim 1, claim 28, or both, comprising at least one of the following:
-
a container; one or more solutions, substances, devices or combinations thereof for collecting a urine comprising sample; one or more solutions, substances, devices or combinations thereof for collecting a plasma comprising sample; one or more solutions, substances, devices or combinations thereof for DNA isolation; one or more solutions, substances, devices or combinations thereof for bisulfite treatment of DNA; one or more solutions, substances, devices or combinations thereof for amplification of bisulfite converted DNA; one or more solutions, substances, devices or combinations thereof for methylation status or methylation pattern determination; and a description for carrying out a method according to one of the preceeding claims.
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-
54. A kit for carrying out a method of claim 53 comprising:
-
a container; one or more solutions, substances, devices or combinations thereof for collecting a urine comprising sample; one or more solutions, substances, devices or combinations thereof for collecting a plasma comprising sample; one or more solutions, substances, devices or combinations thereof for DNA isolation; one or more solutions, substances, devices or combinations thereof for bisulfite treatment of DNA; one or more solutions, substances, devices or combinations thereof for methylation status or methylation pattern determination; and a description for carrying out a method according to one of the preceeding claims.
-
-
55. The kit of claim 54, further comprising:
one or more solutions, substances, devices or combinations thereof for amplification of bisulfite converted DNA.
-
57. The kit of claim 53 or 56, wherein one or more solutions, substances, devices or combinations thereof for amplification of bisulfite converted DNA comprises:
-
a ligase activity, a terminal transferase activity, or both; a polymerase activity; at least one primer; and at least one nucleotide, at least one oligomer, or both.
-
-
58. The kit of claim 57, wherein
the ligase activity is a single stranded DNA ligase; -
the terminal transferase activity is a terminal deoxynucleotidyl transferase; the polymerase activity is a DNA polymerase, a heatstable DNA polymerase, a RNA transcriptase, a RNA transcriptase, in combination with a RNase as an additional enzyme, or a ligase; the primer or primers are random primers, guanin-poor random primers, specific primers, gene specific primers, or extension specific primers; the oligomer is a oligonucleotide or a chimeric oligomer of at least one PNA-monomer and a 5′
or 3′
terminal nucleotide; andcombinations thereof.
-
-
61. The kit of claim 53 or 60, wherein one or more solutions, substances, devices or combinations thereof for bisulfite treatment of DNA comprises:
-
a bisulfite reagent; and a radical scavenger.
-
-
62. The kit of claim 53, wherein one or more solutions, substances, devices or combinations thereof for collecting a plasma comprising sample comprises at least one of the following:
-
a container comprising EDTA; a container comprising negative pressure; a syringe; one or more container suitable for centrifugation; one or more pipets; one or more container suitable for cooling, freezing, storing, transporting, or combinations thereof of the plasma comprising sample; a case report form; and a process checklist.
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-
63. The kit of claim 53, wherein one or more solutions, substances, devices or combinations thereof for collecting a urine comprising sample comprises at least one of the following:
-
a urine collection cup; a pipet; one or more container comprising EDTA suitable for cooling, freezing, storing, transporting, or combinations thereof of the urine comprising sample; a case report form; and a process checklist.
-
-
64. The kit of claim 53, further comprising at least one of the following:
-
one or more solutions, substances, devices or combinations thereof for concentrating a remote sample or at least one component of a remote sample; and one or more solutions, substances, devices or combinations thereof for purifying bisulfite treated DNA.
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2. The method of claim 1, wherein the DNA of the remote sample is characterized in that less than about 5%, less than about 3%, less than about 1%, or less than about 0.1% of the DNA is derived from a defined cell, group of cells, tissue or organ.
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28. A method for amplification of at least one nucleic acid, comprising:
-
providing a nucleic acid sample comprising at least one nucleic acid molecule; treating at least one nucleic acid molecule derived from said sample with an enzyme or reagent which differentiates between methylated bases within said nucleic acid molecule and unmethylated bases within said nucleic acid molecule; extending at least one strand of at least one nucleic acid molecule derived from said sample by at least one nucleotide or PNA-monomer; and amplifying the at least one extended nucleic acid molecule. - View Dependent Claims (29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
-
29. The method of claim 28, wherein the extension is characterized in that the at least one strand of at least one nucleic acid is extended
by one or more single nucleotides or PNA-monomers, by one or more oligonucleotides or PNA-oligomers, by a second nucleic acid derived from the provided nucleic acid sample, or by combinations thereof. -
30. The method of claim 28, wherein the extension is catalyzed template independently.
-
31. The method of claim 28, wherein the extension is catalyzed by means of at least one enzyme selected from the group consisting of:
- a transferase, a transferase transferring phosphorus-containing groups, a nucleotidyltransferase, a DNA nucleotidylexotransferase, terminal deoxynucleotidyl transferase (TdT), a enzyme with ribonucleotide transferase activity, a polyribonucleotide nucleotidyltransferase, a tRNA nucleotidyltransferase, RNA uridylyltransferase, a ligase, a ligase forming phosphoric ester bonds, a DNA ligase, a ATP dependent DNA ligase, a single stranded DNA ligase, a ATP dependent single stranded DNA ligase catalyzing intramolecular circularization, CircLigase ssDNA Ligase.
-
32. The method of claim 28, wherein the enzyme or reagent differentiating between methylated bases and unmethylated bases is a bisulfite reagent.
-
33. The method of claim 28, wherein the provided nucleic acid is at least in part DNA, RNA or PNA.
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34. The method of claim 28, wherein the providing of a nucleic acid sample comprises at least one of the following:
- fragmentation, random fragmentation, fragmentation by mechanical stress, fragmentation by means of an enzyme, fragmentation by means of an nuclease, and fragmentation by means of an restriction endonuclease.
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35. The method of claim 28, wherein the amplifying of at least one extended nucleic acid molecule comprises at least one of the following a polymerase, a heatstable polymerase, nucleotide, oligonucleotide, a ligase, a reverse transcriptase, a RNA polymerase, and an RNase.
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36. The method of claim 28, wherein the amplifying of at least one extended nucleic acid molecule comprises the use of at least one method selected from the group consisting of:
- an amplification method, a PCR method, an isothermal amplification method, a NASBA method, an LCR method, and combinations thereof.
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37. The method of claim 28, wherein the methylation of the provided nucleic acid molecule comprises analysis by at least one method selected from the group consisting of:
- an amplification method, a PCR method, an isothermal amplification method, a NASBA method, an LCR method, a methylation specific amplification method, an MSP (Methylation Specific PCR) method, a nested MSP method, a HeavyMethyl™
method, a detection method, a methylation specific detection method, a bisulfite sequencing method, detection by means of DNA-arrays, detection by means of oligonucleotide microarrays, detection by means of CpG-island-microarrays, detection by means of restriction enzymes, simultaneous methylation specific amplification and detection method, a COBRA method, real-time PCR, HeavyMethyl™
real time PCR method, MSP MethyLight™
method, MethyLight™
method, MethyLight™
Algo™
method, QM method, Headloop MethyLight™
method, HeavyMethyl™
MethyLight™
method, HeavyMethyl™
Scorpion™
method, MSP Scorpion™
method, Headloop Scorpion™
method, methylation sensitive primer extension, and Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) method, and combinations thereof.
- an amplification method, a PCR method, an isothermal amplification method, a NASBA method, an LCR method, a methylation specific amplification method, an MSP (Methylation Specific PCR) method, a nested MSP method, a HeavyMethyl™
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38. The method of claim 28, comprising:
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providing a DNA sample comprising at least one DNA molecule; extending at least one strand of the said provided at least one DNA molecule by at least one single nucleotide or PNA-monomer; treating the extended at least one DNA strand with an enzyme or reagent which differentiates between methylated cytosine within the said DNA molecule and unmethylated cytosine within said DNA molecule; and amplifying at least one treated DNA molecule comprising at least one extended strand.
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39. The method of claim 38, wherein the extending of at least one strand of the provided at least one DNA molecule comprises terminal nucleotidyl transferase and one or more nucleotides.
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40. The method of claim 38, wherein the amplifying of the treated DNA molecule is characterized in that an oligonucleotide or oligomer is at least in part hybridized to the extended portion of the said DNA molecule.
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41. The method of claim 28, comprising:
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providing a DNA sample comprising at least one double stranded DNA molecule or at least two single stranded DNA molecules; treating the provided DNA with an enzyme or reagent which differentiates between methylated cytosine within the said DNA and unmethylated cytosine within said DNA, wherein treated single stranded DNA molecules are provided; extending at least one of the said treated single stranded DNA molecules by at least one oligonucleotide or PNA-oligomer or by at least one additional treated single stranded DNA molecule; and amplifying at least one single stranded DNA molecule after treatment and extension.
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42. The method of claim 41, wherein the extending of at least one treated single stranded DNA molecule comprises a single stranded DNA ligase.
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43. The method of claim 41, wherein the amplifying of the said DNA molecule is characterized in that at least one oligonucleotide or oligomer is at least in part hybridized on the extended portion of the treated single stranded DNA molecule.
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44. The method of claim 41, wherein the treated single stranded DNA molecule is intramolecular ligated during the extension step, and the amplifying is characterized in that at least one oligonucleotide or oligomer hybridizes at an arbitrary site of the circularisized treated single stranded DNA molecule.
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29. The method of claim 28, wherein the extension is characterized in that the at least one strand of at least one nucleic acid is extended
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56. A kit, comprising:
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a container; and one or more solutions, substances, devices or combinations thereof for amplification of bisulfite converted DNA. - View Dependent Claims (59, 60)
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59. The kit of claim 56, further comprising:
a description or manual for carrying out an amplification of bisulfite treated DNA according to one of the proceeding claims.
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60. The kit of claim of claim 56, further comprising:
one or more solutions, substances, devices or combinations thereof for bisulfite treatment of DNA.
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59. The kit of claim 56, further comprising:
-
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65. Use of a method or kit according to one of the preceeding claims for the analysis of at least one DNA methylation status, at least one DNA methylation level, or of at least one DNA methylation pattern.
- View Dependent Claims (66, 67, 68, 69, 70, 71)
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66. Use of a method or kit of claim 65 for identifying an indication-specific target, comprising:
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a) detecting the percentage of DNA fragments characterized by a defined methylation pattern within a mixture of DNA fragments which are derived from a diseased cell, group of cells, tissue or organ; b) detecting the percentage of DNA fragments characterized by a defined methylation pattern within a mixture of DNA fragments which are derived from a healthy cell, group of cells, tissue or organ; and c) defining a indication-specific target based on differences in the percentages of the DNA derived from the diseased cell, group of cells, tissue or organ in comparison to the DNA derived from the healthy cell, group of cell, tissue or organ.
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67. The use of a method or kit of claim 66, wherein the indication-specific target is a DNA section, a RNA molecule, a protein, a peptide or metabolic compound.
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68. The use of a method or kit of claim 67, wherein a per se known modulator of said DNA section, said RNA molecule, said protein, said peptide or said metabolic compound is assigned to the specific indication of the diseased cell, group of cell or tissue.
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69. The use of the modulator assigned according to claim 68 for preparing a pharmaceutical composition in case of a specific indication, or a specific cancer indication.
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70. Use of a method or kit according to claim 65 for at least one of the following with regard to a patient or individual:
- diagnosing a condition, prognosing a condition, predicting a treatment response, diagnosing a predisposition for a condition, diagnosing a progression of a condition, grading a condition, staging a condition, classification of a condition, characterization of a condition, or combinations thereof, wherein the condition is a healthy condition or an adverse event, the adverse event comprises at least one category selected from the group comprising;
undesired drug interactions;
cancer diseases, proliferative diseases or therewith associated diseases;
CNS malfunctions;
damage or disease;
symptoms of aggression or behavioral disturbances;
clinical;
psychological and social consequences of brain damages;
psychotic disturbances and personality disorders;
dementia and/or associated syndromes;
cardiovascular disease of the gastrointestinal tract;
malfunction, damage or disease of the respiratory system;
lesion, inflammation, infection, immunity and/or convalescence;
malfunction, damage or disease of the body as an abnormality in the development process;
malfunction, damage or disease of the skin, of the muscles, of the connective tissue or of the bones;
endocrine and metabolic malfunction, damage or disease; and
headaches or sexual malfunction.
- diagnosing a condition, prognosing a condition, predicting a treatment response, diagnosing a predisposition for a condition, diagnosing a progression of a condition, grading a condition, staging a condition, classification of a condition, characterization of a condition, or combinations thereof, wherein the condition is a healthy condition or an adverse event, the adverse event comprises at least one category selected from the group comprising;
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71. Use of a method or kit according to claim 65 for distinguishing cell types or tissue, or for investigating cell differentiation, wherein condition A and condition B are different cell conditions.
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66. Use of a method or kit of claim 65 for identifying an indication-specific target, comprising:
Specification
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Current AssigneeNew Day Diagnostics LLC
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Original AssigneeEpigenomics AG
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InventorsSledziewski, Andrew Z., Lofton-Day, Catherine, De Vos, Theo, Lograsso, Joe, Schuster, Matthias, Ballhause, Matthias, Maas, Jennifer, Model, Fabian, Liebenberg, Volker, Dietrich, Dimo, Tetzner, Reimo, Berlin, Kurt
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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 CodesC12N 15/1003 Extracting or separating nu...C12Q 1/6806 Preparing nucleic acids for...C12Q 1/6883 for diseases caused by alte...C12Q 2521/131 Terminal transferaseC12Q 2523/125 Bisulfite(s)C12Q 2531/101 Linear amplification, i.e. ...C12Q 2600/154 Methylation markersY10T 436/143333 Saccharide [e.g., DNA, etc.]