Method for determining the presence or absence of methylation in a sample

• US 10,731,215 B2
• Filed: 04/17/2006
• Issued: 08/04/2020
• Est. Priority Date: 04/15/2005
• Status: Active Grant

• Current Assignee: New Day Diagnostics LLC
• Original Assignee: Epigenomics AG
• Inventors: Ballhause, Matthias, Berlin, Kurt, De Vos, Theo, Dietrich, Dimo, Liebenberg, Volker, Lofton-Day, Catherine, Lograsso, Joe, Maas, Jennifer, Model, Fabian, Schuster, Matthias, Sledziewski, Andrew Z., Tetzner, Reimo
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US11/910,887
• Publication Number:
  US 20110059432A1
• Time in Patent Office: 5,223 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/131 Terminal transferase
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2531/101 Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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. Other aspects provide methods for determining the presence or absence of methylation of at least one cytosine, or a series of cytosines in cis, in human DNA of a blood sample, a plasma sample, a serum sample or a urine sample from a human individual.

Claims

1. A method for determining the presence or absence of methylation of at least one cytosine, or a series of cytosines in cis, in human DNA of a blood sample, a plasma sample, a serum sample or a urine sample from a human individual, comprising:

(a) providing the sample comprising human DNA,

(b) isolating human DNA from the sample;

(c) treating the isolated human DNA with bisulfite to produce bisulfite-treated sulfonated DNA;

(d) heating the bisulfite-treated sulfonated DNA of step (c) to a temperature between 85°

Celsius and 100°

Celsius for between 3 minutes and 18 hours, thereby desulfonating the bisulfite-treated sulfonated DNA;

(e) performing methylation specific amplification on the heated DNA of step (d) without any prior desulfonation step comprising an alkaline reagent, wherein the methylation specific amplification comprises steps of denaturation, annealing two or more primers and extension of the two or more primers;

and

(f) detecting the presence or absence of methylation of at least one cytosine, or a series of cytosines in cis when the amplified DNA of step (e) is amplified, thereby determining the presence or absence of methylation of at least one cytosine, or a series of cytosines in cis, in human DNA of the blood sample, the plasma sample, the serum sample or the urine sample from the human individual.

2. The method of claim 1, wherein said sample provided in step (a) is plasma, and the providing of the plasma sample comprises:

(a) obtaining blood from a human individual;

(b) adding EDTA (ethylene-diamine-tetra-acetic acid) to the blood and mixing and centrifuging to obtain plasma comprising human DNA;

(c) transferring the plasma into a new container;

(d) centrifuging the plasma;

(e) transferring the re-centrifuged plasma into a new container;

(f) cooling the plasma to a temperature of 0°

Celsius to 10°

Celsius; and

(g) freezing, storing or transporting the plasma;

wherein all of the steps (a) to (g) are performed within 8 hours.

3. The method of claim 1, wherein the sample provided in step (a) is urine and the providing of the urine sample comprises:

(a) 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 10 seconds to 120 seconds;

(b) collecting voided urine comprising human DNA;

(c) adding EDTA to the urine;

wherein the EDTA has a pH of 5.0, 6.0, 7.0, 7.5, 8.0, 8.5, 9.0, or 10;

(d) cooling the urine to a temperature of 0°

Celsius to 10°

Celsius; and

(e) freezing, storing or transporting the urine wherein steps (b) to (e) are performed within 120 minutes.

4. The method of claim 1, wherein said sample provided in step (a) is divided into different sub-samples subsequent to providing said sample, and/or wherein said sample provided in step (a) or at least one component of said sample is concentrated subsequent to providing said sample.

5. The method of claim 4, wherein the concentration comprises ultrafiltration.

6. The method of claim 1, wherein the isolation of human DNA in step (b) comprises:

treating the sample with a protease;

treating the sample with a chaotropic salt or a detergent;

purifying and washing the human DNA.

7. The method of claim 1, wherein step (c) comprises:

mixing of 10 to 250 μ

l of a solution comprising the human DNA with 45 to 75 μ

l of bisulfite solution, the bisulfite solution having a pH in the range of 5.45 to 5.50 comprising 4.83 to 4.93 mol/l hydrogen sulfite; and

adding 5 to 500 μ

l of an organic radical scavenger solution, the organic radical scavenger solution comprising an organic solvent and 10 to 750 mmol/l of β

-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid.

8. The method of claim 1, wherein after the heating of step (d) and prior to step (e) the heated DNA is cooled to a temperature between 0°

Celsius and 80°

Celsius.