Methods for isolating nucleic acids with size selection

Patent metadata

Field	Value
Publication number	US-12398389-B2
Application number	US-201916969892-A
Country	US
Kind code	B2
Filing date	Feb 15, 2019
Priority date	Feb 15, 2018
Publication date	Aug 26, 2025
Grant date	Aug 26, 2025

Abstract

Disclosed here is a method for isolating nucleic acids from a biological sample, comprising: (a) contacting a first composition comprising nucleic acids obtained from a biological sample with a first matrix under a low-stringency binding condition having less than 1% aliphatic alcohols that binds less than 5% of nucleic acids of shorter than about 118 bp and more than 30% of nucleic acids longer than about 194 bp to a first matrix; and (b) contacting a second composition comprising remainder of the first composition with a second matrix under a high-stringency binding condition having less than 1% aliphatic alcohol that binds more than 70% of nucleic acids longer than about 72 bp and 30% of nucleic acids longer than about 50 bp to the second matrix. Further disclosed is a kit for isolating nucleic acids from a biological sample, comprising (a) a first binding buffer for establishing a low-stringency binding condition having less than 1% aliphatic alcohols that binds less than 5% of nucleic acids shorter than about 118 bp and more than 30% of nucleic acids longer than about 194 bp to a matrix, and (b) a second binding buffer for establishing a high-stringency binding condition having less than 1% aliphatic alcohol that binds more than 70% of nucleic acids longer than about 72 bp and 30% of nucleic acids longer than about 50 bp to the matrix.

First claim

What is claimed is:

1. A method for isolating nucleic acids from a biological sample, comprising:
(a) contacting a first composition comprising nucleic acids in a biological sample with a first solid phase nucleic acid binding matrix to obtain a filtrate and a first eluate, wherein the first composition comprises:
(i) less than 1% aliphatic alcohols,
(ii) about 3.2055 M to about 4.76 M of a chaotropic compound,
(iii) about 0% to about 12.1% of a nitrile compound,
(iv) a pH of about 6.6 to about 7.157,
(v) 0% to about 9% of a detergent, and
(vi) 0% to about 1.2% of a chelating agent;
wherein the first composition permits binding of greater than 30% of nucleic acids longer than about 194 bp to the first matrix, wherein the filtrate comprises remainder nucleic acids passed through the first matrix, and wherein the first eluate comprises nucleic acids bound and released from the first matrix; and

(b) contacting a second composition comprising the filtrate comprising remainder nucleic acids from the first composition with a second solid phase nucleic acid binding matrix to obtain a second eluate, wherein the second composition comprises:
(i) less than 1% aliphatic alcohols,
(ii) about 3.2055 M to about 4.76 M of a chaotropic compound,
(iii) about 20% to about 30% of a nitrile compound,
(iv) a pH of about 4.05 to about 5.5,
(v) 0% to about 9.55% of a detergent, and
(vi) about 0 to about 0.61 mM of a chelating agent;
wherein the second composition binds more than 40% of nucleic acids longer than about 72 bp to the second matrix, wherein the second eluate comprises nucleic acids bound and released from the second matrix; wherein the first and the second solid phase nucleic acid binding matrices comprise siliceous materials, silica gel, glass, glass fiber, or gelatinous silica.

2. The method of claim 1 , further comprising washing the first matrix with a washing buffer after step (a), and eluting nucleic acids from the first matrix with an elution buffer to obtain the first eluate.

3. The method of claim 1 , wherein the second eluate is obtained by washing the second matrix with a washing buffer after the contacting in step (b), and eluting nucleic acids from the second matrix with an elution buffer.

4. The method of claim 1 , further comprising digesting the biological sample with a protease prior to step (a).

5. The method of claim 1 ,
wherein the nitrile compound of the first and/or second composition comprises acetonitrile (ACN), propionitrile (PCN), butyronitrile (BCN), isobutylnitrile (IBCN), or a combination thereof; and
wherein the chaotropic compound of the first and/or second composition comprises guanidine chloride (GnCl), urea, thiourea, guanidine thiocyanate, NaI, guanidine isothiocyanate, D-/L-arginine, a perchlorate or perchlorate salt of Li+, Na+, K+, or a combination thereof.

6. The method of claim 1 , wherein the first composition comprises ACN as the nitrile compound, and about 3.2055 M to about 4 M of GnCl as the chaotropic compound; and wherein the second composition comprises ACN as the nitrile compound and about 3.5 M to about 4.76 M of GnCl as the chaotropic compound.

7. The method of claim 1 , wherein the detergent comprises Triton X-100, Tween 20, N-lauroyl sarcosine, sodium dodecylsulfate (SDS), dodecyldimethylphosphine oxide, sorbitan monopalmitate, decylhexaglycol, 4-nonylphenyl-polyethylene glycol, or a combination thereof; and wherein the chelating compound comprises ethylenediaminetetraccetic (EDTA), ethyleneglycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), citric acid, N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), 2,2′-Bipyridyl, deferoxamine methanesulfonate salt (DFOM), 2,3-Dihydroxybutanedioic acid (tartaric acid), or a combination thereof; and
wherein the first composition and/or the second composition comprises less than 1% of alcohol.

8. The method of claim 1 , wherein the biological sample is a plasma sample from a pregnant woman comprising fetal cfDNA and maternal cfDNA, or a plasma sample from a cancer patient comprising circulating tumor DNA; wherein when the biological sample is a plasma sample from a pregnant woman, fetal fraction of nucleic acids elutable from the second solid phase nucleic acid binding matrix is at least 1% higher than fetal fraction of nucleic acids elutable from the first solid phase nucleic acid binding matrix.

9. The method of claim 1 , wherein step (a) binds more than 40% of nucleic acids longer than about 194 bp to the first solid phase nucleic acid binding matrix, and wherein step (b) binds more than 80% of nucleic acids longer than about 72 bp to the second solid phase nucleic acid binding matrix.

10. The method of claim 1 , wherein the first composition further comprises Tris(hydroxymethyl) aminomethane (Tris-base), a chelating compound, and a detergent.

11. The method of claim 10 , wherein the first composition comprises the biological sample in an amount from 38.9% to about 45.66%.

12. The method of claim 10 , wherein the nitrile compound is acetonitrile, the chelating compound is EDTA, the detergent is Tween 20, and the chaotropic compound is guanidine chloride; wherein the biological sample is a plasma sample from a pregnant woman comprising fetal cell-free DNA (cfDNA) and maternal cfDNA, or a plasma sample from a cancer patient comprising circulating tumor DNA (ctDNA); and wherein:
the method further comprising amplifying the cfDNA or ctDNA prior to step (a) by ligating the cfDNA or the ctDNA to a plurality of DNA adapter molecules, wherein the DNA adapter molecules comprises common forward and reverse primer binding sites, and then amplifying the ligated cfDNA or ctDNA by using forward and reverse primers complementary to the common primer binding sites in the DNA adaptor molecules, or
the method further comprising adding a plurality of DNA base pairs to the cfDNA or ctDNA fragments by tailing PCR to increase the size of the cfDNA or ctDNA fragments prior to step (a).

13. The method of claim 1 , wherein the second composition comprises 2-(N-morpholino) ethanesulfonic acid (MES).

14. The method of claim 13 , wherein the second composition comprises, the MES in an amount from about 4.59 mM to about 38 mM.

Assignees

• Natera Inc

Inventors

• Stray James
• Tong Jason

Classifications

• C12Q1/6853

  using modified primers or templates · CPC title

• C12N15/1017

  by filtration, e.g. using filters, frits, membranes · CPC title

• C12N15/1013 Primary

  by using magnetic beads · CPC title

• C12N15/1006 Primary

  by means of a solid support carrier, e.g. particles, polymers · CPC title