Method and materials for bisulfite conversion of cytosine to uracil

• US 7,371,526 B2
• Filed: 08/26/2004
• Issued: 05/13/2008
• Est. Priority Date: 08/29/2003
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Zon, Gerald, Boyd, Victoria L.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/926,531
• Publication Number:
  US 20050095623A1
• Time in Patent Office: 1,356 Days
• Field of Search: 435/6, 435/810, 536/23.1, 436/808
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

The invention provides methods and materials for conversion of cytosine to uracil. In some embodiments, a nucleic acid, such as gDNA, is reacted with at least one bisulfite salt having the formula X⁺HSO₃⁻ or Y⁺²(HSO₃³¹)₂; wherein X⁺ is ammonium ion, a tetraalkyl ammonium ion, or a group 1A ion other than sodium; and Y⁺² is a group 2A ion or a group 7B ion; under conditions effective to convert at least one cytosine nucleobase to a uracil nucleobase. In some embodiments, X⁺ comprises at least one of lithium ion, potassium ion, ammonium ion, tetraalkylammonium ion, magnesium ion, manganese ion and calcium ion. In some embodiments, the reacting is performed optionally in the presence of a polyamine catalyst and/or a quaternary amine catalyst. Also provided are kits that can be used to carry out methods of the invention.

Claims

1. In a method of converting at least one cytosine nucleobase to a uracil nucleobase in a nucleic acid wherein the nucleic acid is treated with bisulfite ion, the improvement comprising:

performing said treatment of the nucleic acid with bisulfite ion in the presence of Mg²⁺ ion.

2. The method of claim 1, wherein said treatment is performed in a solution containing magnesium bisulfite in a concentration of from about 0.5M to about 2.5M.

3. The method of claim 2, wherein said solution contains magnesium bisulfite in a concentration of 1M to 2M.

4. The method of claim 1, wherein said treatment is performed at 40 degrees to 60 degrees for 4 to 15 hours.

5. The method of claim 1, wherein said nucleic acid is gDNA.

6. The method of claim 1, wherein said nucleic acid is obtained from microorganisms.

7. The method of claim 2, wherein said nucleic acid is obtained from DNA or RNA viruses.

8. The method of claim 1, further comprising treating the product of said reaction of said nucleic acid and said bisulfite ion with a base.

9. The method of claim 8, wherein said base comprises NaOH.

10. A kit for use in converting at least one cytosine nucleobase to a uracil nucleobase in a nucleic acid wherein the nucleic acid is treated with bisulfite ion, the improvement comprising:

performing said treatment of the nucleic acid with bisulfite ion in the presence of Mg²⁺ ion.

11. The kit of claim 10, wherein the bisulfite ion and the Mg²⁺ ion are present as magnesium bisulfite.

12. The kit of claim 10, wherein said treatment is performed in a solution containing magnesium bisulfite in a concentration of from about 0.5M to about 2.5M.

13. The kit of claim 10, wherein said solution contains magnesium bisulfite in a concentration of 1M to 2M.

14. The kit of claim 10, wherein said treatment is performed at 40 degrees to 60 degrees for 4 to 15 hours.

15. The kit of claim 10, wherein said nucleic acid is gDNA.

16. The kit of claim 10, wherein said nucleic acid is obtained from microorganisms.

17. The kit of claim 10, wherein said nucleic acid is obtained from DNA or RNA viruses.

18. The kit of claim 10, further comprising treating the product of said reaction of said nucleic acid and said bisulfite ion with a base.

19. The kit of claim 18, wherein said base comprises NaOH.