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

• US 9,453,257 B2
• Filed: 02/14/2014
• Issued: 09/27/2016
• 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: Schmidt, Karsten E, Hoyal-Wrightson, Carolyn R., Braun, Andreas
• Primary Examiner(s): PRIEST, AARON A
• Application Number:
  US14/180,810
• Publication Number:
  US 20140255943A1
• Time in Patent Office: 956 Days
• Field of Search: None
• US Class Current: 1/1
• 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: 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 selectively amplifying cell-free target nucleic acid from a biological sample containing a mixture of the target nucleic acid and non-target nucleic acid and for specifically amplifying a nucleic acid region of interest in the target nucleic acid, comprising:

a) mixing said biological sample with a 5′

adapter molecule and a 3′

adapter molecule, wherein;

(i) the target nucleic acid in the biological sample is about 1200 base pairs or less and comprises double-stranded, blunt-ended nucleic acid fragments with 5′

phosphorylated ends, wherein the fragments originate from normal programmed cell death, induced programmed cell death, autoimmune disease, septic shock, malignant neoplasms, non-malignant neoplasms, inflamed tissue, diseased tissue, an allograft, a fetus or a placenta,

(ii) the non-target nucleic acid in the biological sample is greater than 1200 base pairs and more abundant than the target nucleic acid, and

(iii) the 3′

adapter molecule is complementary to the 3′

end of the 5′

adapter molecule, thereby generating a double-stranded adapter complex comprising a 5′

adapter molecule strand and a 3′

adapter molecule strand, wherein the complex is blunt-ended at one end of the double-stranded complex and selectively ligates to the target nucleic acid;

b) without subjecting the mixture of a) to a process that generates blunt ends, introducing a ligase to the mixture of a), whereby the 5′

adapter strand of the double-stranded adapter complex generated in a) ligates, at the blunt end of the complex, to the target nucleic acid fragments at each of the 5′

phosphorylated ends, thereby generating a ligated sample comprising double-stranded adapter complex ligated to the 5′

phosphorylated ends of the target nucleic acid fragments;

c) heating the ligated sample of b) to release the 3′

adapter strands of the double-stranded adapter complex ligated to the 5′

phosphorylated ends of the target nucleic acid fragments, thereby generating products comprising single-stranded 5′

adapter strands comprising 5′

protruding ends at each end of the target nucleic acid;

d) adding a polymerase to fill in the single-stranded 5′

protruding ends of the 5′

adapter strands in the products generated in c);

e) adding 5′

adapter primers that hybridize to the filled in 5′

ends of the products of d) and amplifying the products of d) under conditions comprising short extension times of up to about 10 seconds, whereby the target nucleic acid is selectively amplified and enriched; and

f) performing an additional amplification step after e), wherein the amplification is performed using a target-specific PCR primer and whereby the nucleic acid region of interest is specifically amplified in the target nucleic acid.

2. The method of claim 1, wherein a dideoxy-nucleotide is incorporated into the 3′

position of the 3′

adapter molecule.

3. The method of claim 1, wherein the 5′

adapter molecule of a) is bound to a solid support and whereby b) comprises ligated sample that is bound to the solid support.

4. The method of claim 3, wherein a spacer arm is introduced between the 5′

adapter molecule at the 5′

end and the solid support.

5. The method of claim 3, wherein the solid support-bound ligated sample products are combined with ligated sample that is not bound to the solid support prior to amplification step e).

6. The method of claim 1, comprising extracting the target nucleic acid from the biological sample prior to a).

7. The method of claim 6, wherein the extraction comprises:

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

(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 a 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;

(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.

8. The method of claim 1, wherein the target nucleic acid comprises oligonucleosomes.

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

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

11. The method of claim 1, wherein the sample is plasma.

12. The method of claim 1, wherein the sample is serum.

13. The method of claim 1, wherein the sample is urine.