Nucleic acid preparation compositions and methods

• US 8,771,948 B2
• Filed: 04/01/2010
• Issued: 07/08/2014
• Est. Priority Date: 04/03/2009
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Wisniewski, Michele E., Kwong, William H., Mohsenian, Firouz, Ding, Jian-Hua
• Primary Examiner(s): Martinell, James
• Application Number:
  US13/262,624
• Publication Number:
  US 20120178918A1
• Time in Patent Office: 1,559 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/137 Chromatographic separation
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Provided herein are methods and compositions to extract and enrich by, physical separation or amplification, relatively short nucleic acids from a nucleic acid composition containing a high background of longer nucleic acids (e.g., host or maternal nucleic acids; genomic nucleic acid and the like).

Claims

1. A method for enriching relatively short nucleic acid from cell-free nucleic acid, which comprises:

(a) contacting cell-free nucleic acid from a substantially cell-free biological composition with a solid phase under association conditions, wherein;

(i) the nucleic acid of the biological composition comprises relatively short nucleic acid and relatively long nucleic acid,

(ii) the relatively short nucleic acid is 300 base pairs or less,

(iii) the relatively long nucleic acid is larger than 300 base pairs, and

(iv) the association conditions do not include polyethylene glycol;

whereby the relatively short nucleic acid and the relatively long nucleic acid are associated with the solid phase;

(b) introducing the solid phase after (a) to dissociation conditions that comprise a volume exclusion agent and a salt, wherein;

(i) the salt is not a chaotropic salt, and

(ii) the relatively short nucleic acid preferentially dissociates from the solid phase under the dissociation conditions as compared to the relatively long nucleic acid, thereby yielding dissociated nucleic acid; and

(c) separating the dissociated nucleic acid from the solid phase, whereby the relatively short nucleic acid is enriched in the dissociated nucleic acid relative to in the cell-free nucleic acid from the substantially cell-free biological composition.

2. The method of claim 1, wherein the substantially cell-free biological composition is from a pregnant female.

3. The method of claim 2, wherein the substantially cell-free biological composition is blood serum.

4. The method of claim 2, wherein the substantially cell-free biological composition is blood plasma.

5. The method of claim 2, wherein the substantially cell-free biological composition is urine.

6. The method of claim 1, wherein the solid phase further comprises an agent that confers a paramagnetic property to the solid phase.

7. The method of claim 1, wherein the solid phase has a net charge.

8. The method of claim 1, wherein the dissociated nucleic acid comprises deoxyribonucleic acid (DNA).

9. The method of claim 1, wherein the association conditions comprise a C1-C6 alkyl alcohol.

10. The method of claim 9, wherein the alcohol is ethanol.

11. The method of claim 1, wherein the association conditions comprise a salt.

12. The method of claim 11, wherein the salt is a chaotropic salt, an ionic salt or combination thereof.

13. The method of claim 1, wherein the relatively short nucleic acid is about 200 base pairs or less.

14. The method of claim 13, wherein the relatively short nucleic acid is about 50 to about 180 base pairs.

15. The method of claim 1, which further comprises washing the solid phase after (a).

16. The method of claim 1, wherein the salt is an ionic salt.

17. The method of claim 16, wherein the ionic salt is sodium chloride.

18. The method of claim 1, wherein the volume exclusion agent comprises a polyalkyl alcohol, dextran, Ficoll, polyvinyl pyrrolidone or combination thereof.

19. The method of claim 18, wherein the polyalkyl alcohol is a polyethylene glycol (PEG).

20. The method of claim 19, wherein the PEG is PEG-8000.

21. The method of claim 19 wherein the dissociation conditions comprise about 10% PEG.

22. The method of claim 1, wherein the relatively short nucleic acid preferentially dissociates from the solid phase under the dissociation conditions as compared to the relatively long nucleic acid at a ratio of about 1.05 to about 5 relatively short nucleic acid to relatively long nucleic acid.

23. The method of claim 1, wherein the relatively short nucleic acid is enriched about 10% to about 45% in the dissociated nucleic acid relative to in the cell-free nucleic acid from the substantially cell-free biological composition.