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

• US 10,662,421 B2
• Filed: 08/18/2016
• Issued: 05/26/2020
• 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): Priest, Aaron A
• Application Number:
  US15/240,692
• Publication Number:
  US 20170029807A1
• Time in Patent Office: 1,377 Days
• Field of Search: None
• 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 low molecular weight apoptotic DNA from a cell-free DNA sample comprising a mixture of low molecular weight DNA and high molecular weight DNA, which comprises:

(a) mixing the sample, guanidine (iso)thiocyanate or sodium perchlorate, and a silica solid support comprising silica capable of reversibly binding DNA, thereby forming a first binding solution, wherein the guanidine (iso)thiocyanate or sodium perchlorate is present at 1-4M, at which the high molecular weight DNA selectively adsorbs to the solid support;

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

(c) mixing the fraction separated from the silica solid support in (b) with additional guanidine (iso)thiocyanate or sodium perchlorate, whereby the guanidine (iso)thiocyanate or sodium perchlorate is present at a higher concentration than that of the first binding solution in (a);

(d) introducing additional silica solid support to the mixture in (c), thereby forming a second binding solution, wherein the low molecular weight DNA selectively adsorbs to the additional solid support; and

(e) separating the additional silica solid support from the second binding solution, thereby extracting low molecular weight DNA from the sample, wherein the low molecular weight DNA comprises less than 1200 base pairs and the high molecular weight DNA comprises 1200 or more base pairs.

2. The method of claim 1, wherein the sample is blood serum, blood plasma or urine.

3. The method of claim 1, further comprising eluting the adsorbed DNA from the separated solid support in (e).

4. The method of claim 2, wherein the blood serum, blood plasma or urine is from a pregnant human.

5. The method of claim 1, wherein the low molecular weight DNA is of fetal origin.

6. The method of claim 1, wherein the high molecular weight DNA is of maternal origin.

7. The method of claim 1, wherein the silica solid support comprises a silica coated magnetic bead.

8. The method of claim 1, wherein the guanidine (iso)thiocyanate or sodium perchlorate of (a) is present at a concentration in the range of 10% to 30% weight and the guanidine (iso)thiocyanate or sodium perchlorate of (c) is present at a concentration in the range of 20% to 60% weight.