Bisulfite conversion of DNA

• US 8,257,950 B2
• Filed: 06/13/2011
• Issued: 09/04/2012
• Est. Priority Date: 06/19/2000
• Status: Expired due to Fees

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Epigenomics AG
• Inventors: Berlin, Kurt, Ballhause, Matthias, Cardon, Karen
• Primary Examiner(s): Martinell, James
• Application Number:
  US13/159,323
• Publication Number:
  US 20110250601A1
• Time in Patent Office: 449 Days
• Field of Search: None
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention provides an improved method for the bisulfite conversion of DNA, and facilitates the analysis of cytosine methylation of genomic DNA. Novel combinations of denaturing solvents, new reaction conditions and new purification methods provide surprisingly efficacious methods for bisulfite conversion of DNA relative to prior art methods. The converted DNA may subsequently be analyzed by many different methods.

Claims

1. A method for bisulfite conversion of DNA, comprising reacting isolated genomic DNA with a bisulfite reagent, wherein the reaction is conducted at a temperature in the range of 0-80°

C., and wherein the reaction temperature is increased at least once to a range of about 85-100°

C. for a duration of between about 15 seconds and 6 minutes during the course of the conversion.

2. The method according to claim 1, wherein the temperature is increased from 2 to 5 times.

3. The method according to claim 1, wherein the reaction temperature is increased to about 90 to 98°

C.

4. The method according to claim 1, wherein the converted DNA is purified via magnetic particles.

5. The method according to claim 1, wherein the DNA is from tissue samples or bodily fluids.

6. A method for at least one of diagnosis and prognosis of an adverse event for patients or individuals, comprising use of a method according to claim 1, wherein the adverse event is at least one event selected from the category group consisting of:

undesired drug interactions;

cancer diseases;

CNS malfunctions, damage or disease;

symptoms of aggression or behavioral disturbances;

clinical, psychological and social consequences of brain damage;

psychotic disturbances and personality disorders;

dementia;

cardiovascular disease, malfunction and damage;

malfunction, damage or 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;

headaches or sexual malfunction.

7. A method for distinguishing cell types or tissues, or for investigating cell differentiation, comprising use of a method according to claim 1.