Bisulfite method

• US 7,262,013 B2
• Filed: 08/26/2004
• Issued: 08/28/2007
• 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): WHISENANT, ETHAN C
• Application Number:
  US10/926,534
• Publication Number:
  US 20050079527A1
• Time in Patent Office: 1,097 Days
• Field of Search: 435/6, 536/22.1, 536/23.2, 536/24.3, 536/23.1
• US Class Current: 435/6.12
• 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 2523/125 Bisulfite(s)

Abstract

The invention provides methods for purifying bisulfite-treated nucleic acid samples.

Claims

1. A method for the purification of a bisulfite-treated nucleic acid comprising the steps of:

(a) providing a liquid comprising a bisulfite-treated nucleic acid in a size-exclusion device comprising a porous, size-discriminating barrier;

(b) washing said nucleic acid in said size-exclusion device by passing liquid through said barrier while said imcleic acid is retained by said barrier;

(c) treating said washed nucleic acid with a base in said size-exclusion device to increase the pH; and

(d) optionally washing said nucleic acid resulting from step (c), resulting in a purified bisulfite-treated nucleic acid.

2. The method of claim 1 wherein said step (d) is performed in said size-exclusion device.

3. The method of claim 1 wherein said porous, size-discriminating barrier has a 300K cut off.

4. The method of claim 1 wherein said porous, size-discriminating barrier has a 30K cut off.

5. The method of claim 1 wherein said porous, size-discriminating barrier has a 100K cut off.

6. A method of converting one or more cytosine bases to uracil bases in a nucleic acid, the method comprising:

contacting the nucleic acid with a bisulfite conversion reagent, thereby forming a bisulfite-treated nucleic acid,

washing the treated nucleic acid in a size-exclusion device comprising a porous, size-discriminating barrier that allows residual bisulfite conversion reagent to pass through while retaining the bisulfitetreated nucleic acid, and

subjecting the retained bisulfite-treated nucleic acid to basic conditions comprising a pH of 10 or above, during or after said washing, to form a nucleic acid in which at least one cytosine base has been converted to a uracil base.

7. The method of claim 6 wherein the porous, size-discrimination barrier comprises a membrane.

8. The method of claim 6 wherein said barrier has a 300K cut off.

9. The method of claim 6 wherein said barrier has a 100K cut off.

10. The method of claim 6 wherein said barrier has a 30K cut off.

11. The method of claim 6 wherein said washing is performed under pressure.

12. The method of claim 6 wherein said washing comprises centrifugation of the device.

13. The method of claim 6 wherein said bisulfite conversion reagent comprises sodium bisulfite.

14. The method of claim 6 wherein said bisulfite conversion reagent comprises magnesium bisulfite.

15. The method of claim 6 wherein said basic conditions comprise 0.1M NaOH.