Systems and methods for isolating nucleic acids

• US 10,640,808 B2
• Filed: 03/13/2014
• Issued: 05/05/2020
• Est. Priority Date: 03/13/2013
• Status: Active Grant

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Inventors: Gundling, Gerard, Kowal, Robert, Granados, Edward, Solomon, Natalie, Mak, Wai-Bing, Szostak, Magdalena
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US14/209,225
• Publication Number:
  US 20140272968A1
• Time in Patent Office: 2,245 Days
• CPC Class Codes: C12N 1/06 Lysis of microorganisms
• 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

The present disclosure relates to systems and methods for nucleic acid isolation. In particular, the present disclosure provides systems and methods for isolating low molecular weight circulating nucleic acids from bodily fluids (e.g., plasma).

Claims

1. A method of isolating nucleic acids, comprising:

a) contacting a biological sample comprising cells with a lysis buffer comprising greater than 35% ethanol by volume;

b) contacting said sample of step a) with a solid support that binds cell-free nucleic acid wherein said solid support is a magnetic particle wherein said magnetic particle is coated or functionalized with material that enhances nucleic acid binding;

c) washing said solid support of step b) with a wash buffer; and

d) eluting nucleic acid from said solid support to generate isolated nucleic acid.

2. The method of claim 1, wherein said ethanol is present in said lysis buffer at a concentration of approximately 40% to 60%.

3. The method of claim 1, wherein said ethanol is present in said lysis buffer at a concentration of approximately 45% to 55%.

4. The method of claim 1, wherein said ethanol is present in said lysis buffer at a concentration of approximately 50%.

5. The method of claim 1, wherein said nucleic acid is a circulating DNA.

6. The method of claim 5, wherein said DNA is less than 1000 bases in length.

7. The method of claim 5, wherein said DNA is less than 500 bases in length.

8. The method of claim 5, wherein said DNA is less than 200 bases in length.

9. The method of claim 1, wherein said sample is selected from the group consisting of blood, blood products, serum, and urine.

10. The method of claim 1, wherein said sample is from a subject and the presence of said nucleic acid in said sample is indicative of a disease state in said subject.

11. The method of claim 10, wherein said disease state is cancer.

12. The method of claim 1, further comprising the step of analyzing said sample for the presence of said nucleic acid.

13. The method of claim 12, wherein said analyzing comprises performing a nucleic acid detection assay selected from the group consisting of an amplification assay, a hybridization assay, a methylation status detection assay, and a sequencing assay.

14. The method of claim 13, wherein said amplification is real time PCR and said methylation status detection assay is methylation specific PCR or heavy methyl PCR.

15. A composition, comprising:

a circulating DNA;

a magnetic particle wherein said magnetic particle is coated or functionalized with material that enhances nucleic acid binding; and

a lysis buffer comprising 35% or more ethanol by volume in a mixture with a biological sample comprising one or more cells.

16. The composition of claim 15, wherein said ethanol is present in said lysis buffer at a concentration of approximately 45% to 55%.