Methods and compositions for the extraction and amplification of nucleic acid from a sample

• US 8,679,741 B2
• Filed: 05/30/2007
• Issued: 03/25/2014
• Est. Priority Date: 05/31/2006
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Hoyal-Wrightson, Carolyn R., Braun, Andreas, Schmidt, Karsten E.
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US12/301,985
• Publication Number:
  US 20100297710A1
• Time in Patent Office: 2,491 Days
• Field of Search: None
• US Class Current: 435/6
• 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: C12N 15/1013 by using magnetic beads
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium

Abstract

Provided herein are methods, compositions and kits to extract and relatively enrich by physical separation or amplification short base pair nucleic acid in the presence of a high background of genomic material (e.g., host or maternal nucleic acids).

Claims

1. A method for extracting a target nucleic acid from a cell-free biological sample, which comprises:

(a) mixing a cell-free biological sample, a chaotropic salt and a solid support capable of reversibly binding nucleic acid, wherein;

the sample comprises target nucleic acid and non-target nucleic acid,

the target nucleic acid and the non-target nucleic acid are both DNA or both RNA,

the size of the target nucleic acid is less than the size of the non-target nucleic acid, and

the chaotropic salt is present at a concentration at which the non-target nucleic acid binds to the solid support and substantially no target nucleic acid adsorbs to the solid support, whereby the non-target nucleic acid is bound to the solid support in a first binding solution, wherein the first binding solution does not include alcohol and chloroform;

(b) separating the solid support from the first binding solution, thereby yielding a fraction separated from the solid support;

(c) mixing the fraction separated from the solid support with additional chaotropic salt and an additional solid support capable of reversibly binding nucleic acid, wherein;

the chaotropic salt is present at a concentration at which the target nucleic acid binds to the solid support, whereby the target nucleic acid is bound to the solid support in a second binding solution, wherein the second binding solution does not include alcohol and chloroform and wherein the type of the additional solid support is the same as the type of the solid support in (a);

(d) separating the solid support from the second binding solution; and

(e) eluting the target nucleic acid from the solid support after (d), whereby the target nucleic acid is extracted from the cell-free biological sample.

2. The method of claim 1, wherein the cell-free biological sample is blood serum.

3. The method of claim 1, wherein the cell-free biological sample is blood plasma.

4. The method of claim 1, wherein the chaotropic salt of (a) is the same as the chaotropic salt of (c).

5. The method of claim 2, wherein the blood serum is from a pregnant human.

6. The method of claim 3, wherein the blood plasma is from a pregnant human.

7. The method of claim 1, wherein the target nucleic acid comprises at least 25 base pairs, but less than 1200 base pairs.

8. The method of claim 1, wherein the target nucleic acid comprises between about 200 base pairs to about 600 base pairs.

9. The method of claim 1, wherein the target nucleic acid is selected from the group consisting of DNA, RNA, mRNA, oligonucleosomal, mitochondrial, epigenetically modified, single-stranded, double-stranded and nucleic acid including unique DNA sequences.

10. The method of claim 1, wherein the target nucleic acid is an apoptotic product.

11. The method of claim 1, wherein the target nucleic acid is of fetal origin.

12. The method of claim 1, wherein the non-target nucleic acid is of maternal origin.

13. The method of claim 1, wherein the chaotropic salt is at least one compound selected from the group consisting of guanidine (iso)thiocyanate, sodium iodide, sodium perchlorate, potassium iodide, sodium thiocyanate, urea, ammonium perchlorate, and combinations thereof.

14. The method of claim 1, wherein the solid support is selected from the group consisting of paramagnetic microparticles, silica gel, silica particles, controlled pore glass, magnetic beads, biomagnetic separation beads, microspheres, divinylbenzene (DVB) resin, cellulose beads, capillaries, filter membranes, columns, nitrocellulose paper, flat supports, glass surfaces, metal surfaces, plastic materials, multiwell plates or membranes, wafers, combs, pins and needles.

15. The method of claim 1, wherein the chaotropic salt of step c) is added to yield a second binding solution with a chaotropic salt concentration greater than 10% weight per volume.

16. The method of claim 15, wherein the chaotropic salt of step c) is added to yield a second binding solution with a chaotropic salt concentration in the range of 20 to 60% weight per volume.

17. The method of claim 1, wherein the target nucleic acid and the non-target nucleic acid are both DNA.

18. The method of claim 11, wherein the target nucleic acid and the non-target nucleic acid are both DNA.

19. The method of claim 1, wherein the alcohol is phenol.

20. The method of claim 1, wherein prior to eluting the target nucleic acid from the solid support, the solid support-target nucleic acid complex is washed with one or more solutions comprising alcohol and water.