COMPOSITIONS AND METHODS FOR ISOLATION OF CELL-FREE DNA
Provided herein are compositions and methods for isolating cell-free nucleic acid, e.g., cell-free DNA, from a sample. In particular embodiments, provided herein are compositions and methods using anti-dsDNA antibodies for isolating cell-free DNA from a sample, and for providing a sample of isolated cell-free DNA, e.g., for a nucleic acid assay. In particular embodiments, the technology relates to providing cell-free DNA from a maternal sample that is enriched for fetal cell-free fetal DNA.
1 - 49 . (canceled)
50 . A method, comprising:
a) mixing blood or blood product sample from a subject with a solution comprising an exogenous anti-dsDNA antibody that has reactivity to both single-stranded and double-stranded DNA to form a solution comprising antibody-DNA complexes;
b) combining the solution comprising the antibody-DNA complexes with an antibody-binding reagent to produce bound antibody-DNA complexes; and
c) separating the bound antibody-DNA complexes from the solution to provide isolated antibody-DNA complexes.
51 . The method of claim 50 , wherein the antibody-binding reagent comprises a protein selected from protein A, protein G, protein A/G, and protein L.
52 . The method of claim 50 , wherein the antibody-binding reagent comprises one or more beads.
53 . The method of claim 52 , wherein the one or more beads comprises one or more paramagnetic beads.
54 . The method of claim 50 , wherein the blood product comprises serum or plasma.
55 . The method of claim 50 , further comprising combining DNA from the isolated antibody-DNA complexes with a solution comprising a buffer.
56 . The method of claim 55 , wherein prior to said combining, the DNA is released from the isolated antibody-DNA complexes.
57 . The method of claim 55 , wherein combining the DNA from the isolated antibody-DNA complexes with the solution comprising a buffer comprises combining isolated antibody-DNA complexes with the solution comprising a buffer.
58 . The method of claim 55 , wherein the solution comprising a buffer further comprises one or more of:
a) a detergent;
b) a preservative;
c) a protease inhibitor; and
d) a nuclease inhibitor.
59 . The method of claim 55 , wherein the solution comprising a buffer comprises a reaction mixture.
60 . The method of claim 59 , wherein the reaction mixture comprises at least one nucleic acid-modifying enzyme selected from a nucleic acid polymerase, a nuclease, and a ligase.
61 . The method of claim 50 , wherein the subject is a human subject.
62 . The method of claim 61 , wherein the human subject is a pregnant subject or a subject suspected of having a tumor.
63 . The method of claim 50 , wherein the isolated antibody-DNA complexes comprise cell-free DNA.
64 . The method of claim 50 , wherein the isolated antibody-DNA complexes comprise fetal DNA.
65 . A kit or system for isolating DNA from a blood or blood product sample from a human subject, the kit or system comprising:
a) an exogenous anti-dsDNA antibody that has reactivity to both single-stranded and double-stranded DNA; and
b) antibody-binding reagent comprising one or more beads,
wherein the kit or system further comprises one or more of:
c) a nucleic acid modifying enzyme; and
d) one or more oligonucleotides that comprise sequence complementary to human DNA.
66 . The kit or system of claim 65 , wherein the antibody-binding reagent comprises a protein selected from protein A, protein G, protein A/G, and protein L.
67 . The kit or system of claim 65 , wherein the one or more oligonucleotides comprise one or more of:
i) a molecular inversion probe (MIP);
ii) an amplification primer;
iii) a hybridization probe;
iv) a flap oligonucleotide; and
v) an invasive oligonucleotide.
68 . The kit or system of claim 65 , further comprising one or more of:
e) a buffer;
f) a salt;
g) a detergent;
h) a preservative;
i) a protease inhibitor; and
j) a nuclease inhibitor.