Method for providing DNA fragments derived from an archived sample

• US 8,962,246 B2
• Filed: 12/02/2013
• Issued: 02/24/2015
• Est. Priority Date: 09/30/2004
• Status: Active Grant

• Current Assignee: Epigenomics AG
• Original Assignee: Epigenomics AG
• Inventors: Ballhause, Matthias, Berlin, Kurt, Dietrich, Dimo, Lukas, Antje Kluth, Schuster, Matthias, Wagner, Ute, Wasserkort, Reinhold, Ziebarth, Heike
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/094,667
• Publication Number:
  US 20140087449A1
• Time in Patent Office: 449 Days
• Field of Search: 435/6.1, 536/23.1
• US Class Current: 435/6.1
• 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 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2527/149 Concentration of an enzyme

Abstract

Aspects of the present invention relate to compositions and methods for providing DNA fragments from an archived sample (e.g., paraffin-embedded and/or fixed-tissue biopsies, etc.). Particular aspects provide methods whereby high yields of DNA are isolated as well as a substantial portion of the DNA consists of long DNA fragments, and where the isolated genomic DNA is free of associated or cross-linked contaminants like proteins, peptides, amino acids or RNA. The methods are facile, cost-effective, and are characterized by high reproducibility and reliability. Particular aspects provide methods for providing DNA fragments derived from an archived sample, wherein the yield of DNA before, for example, an amplification step is at least 20%, and amplicons up to a length of about 1,000 base pairs are amplifiable.

Claims

1. A method for providing DNA fragments derived from an archived sample, comprising the steps of

a) contacting an archived sample comprising DNA with a protease to provide for an amount of protease-treated DNA;

b) reacting the protease treated DNA of method step a) with a bisulfite reagent without prior DNA extraction, wherein the reaction is carried out in an organic solvent; and

c) purifying the bisulfite-treated DNA.

2. The method of claim 1, wherein method step a) is preceded by a step of paraffin removal.

3. The method of claim 2, wherein paraffin removal is achieved by dissolving the paraffin in an organic solvent.

4. The method of claim 3, wherein the organic solvent is limonene, xylene or a mixture of limonene and xylene.

5. The method according to claim 1, wherein the protease is a serin protease, a thiol protease, a carboxy protease, a metalloprotease, proteinase K or any mixture of said proteases.

6. The method of claim 5, wherein the protease is proteinase K.

7. The method of claim 1, wherein the lysis contacting in method step a) comprises:

(i) addition of 50 μ

l to 1000 μ

l lysis buffer to 1 to 10 deparaffinated, formalin-fixed tissue sections or an equal amount of a deparaffinated formalin-fixed biopsy, wherein the lysis buffer has a pH 8.0 and comprises 50 mM tris-hydroxymethyl-amin-methan, 1 mM EDTA and 0.5% v/v Tween;

(ii) incubation at 40 to 75°

C. for 5 to 20 minutes;

(iii) addition of 5 to 40 μ

l proteinase K solution comprising 30 mg/ml proteinase K; and

(iv) incubation at 40 to 70°

C. for at least 2.5 hours.

8. The method of claim 1, wherein method step b) is carried out in the presence of dioxane.

9. The method of claim 8, wherein the concentration of dioxane is 22 to 28% and the concentration of bisulfite is 3.3 to 3.6 M.

10. The method of claim 1, wherein 6-hydroxy, 2,5,7,8-tetramethylchromane 2-carboxylic acid is used in method step b) as a radical scavenger.

11. The method according to claim 8, wherein 6-hydroxy, 2,5,7,8-tetramethylchromane 2-carboxylic acid is used as radical scavenger.

12. The method according to claim 9, wherein 6-hydroxy, 2,5,7,8-tetramethylchromane 2-carboxylic acid is used as radical scavenger.

13. The method according to claim 1, wherein method step b) is conducted at a temperature of 60°

C. for 4 to 6 hours.

14. The method according to claim 1, wherein the reaction temperature during method step b) is between 57 and 65°

C., interspersed by 1 to 10 temperature increases to 85°

C., wherein the temperature increase

(i) lasts 20 to 50 seconds for a reaction volume of 20 μ

l;

(ii) lasts 1.5 to 3 minutes for a reaction volume of 100 μ

l; and

(iii) lasts 2 to 4 minutes for a reaction volume of 600 μ

l.

15. The method of claim 1, wherein ultrafiltration is used for purifying the DNA in step c).