Acrylamide copolymerization for sequestration and production of single-stranded nucleic acid
Provided herein, in some embodiments, are methods, compositions and kits for large-scale production of long single-stranded DNA in solution.
1. A method of producing single-stranded deoxyribonucleic acid (ssDNA), comprising:
(a) combining in solution acrylamide-labeled double-stranded deoxyribonucleic acid (dsDNA) and acrylamide (AA) monomers to form copolymer-linked dsDNA in solution;
(b) dehybridizing the copolymer-linked dsDNA in solution to produce free ssDNA and DNA-containing copolymer; and
(c) separating the free ssDNA from the solution of (b).
2. The method of claim 1 , wherein separating the free ssDNA from the solution of (b) comprises adding methanol to the solution of (b) to selectively precipitate the DNA-containing copolymer.
3. The method of claim 1 , wherein the length of the dsDNA is 100-10,000 nucleotide base pairs.
4. The method of claim 1 , wherein the concentration of acrylamide monomers in the solution is 10-100 mg/ml.
5. The method of claim 1 , wherein the concentration of acrylamide-labeled dsDNA in the solution is 0.01-10 mg/ml.
6. The method of claim 1 , wherein the solution comprises ammonium persulfate (APS) and tetramethylethylenediamine (TEMED).
7. The method of claim 6 , wherein the solution comprises ammonium persulfate at a concentration of 0.01 wt %-0.05 wt % and/or wherein solution comprises TEMED at a concentration of 0.01 wt %-0.05 wt %.
8. The method of claim 1 , wherein the solution further comprises acrylate (A).
9. The method of claim 8 , wherein the ratio of AA:A is 50:1 to 200:1.
10. The method of claim 8 , wherein the copolymer-linked dsDNA comprises a linear copolymer.
11. The method of claim 1 , wherein the solution comprises ammonium persulfate at a concentration of 0.0005 wt %-0.002 wt % and/or tetramethylethylenediamine (TMED) at a concentration of 0.0005 wt %-0.002 wt %.
12. The method of claim 1 , wherein the solution further comprises a crosslinking agent.
13. The method of claim 12 , wherein the crosslinking agent comprises bisacrylamide (BAA), piperazine diacrylamide, N,N′-Hexamethylenebis(methacrylamide), N,N′-ethylenebis(acrylamide), or combinations thereof.
14. The method of claim 13 , wherein the BAA is N,N-methylenebisacrylamide.
15. The method of claim 13 , wherein the ratio of AA:BAA in the solution is 9:1 to 1:1.
16. The method of claim 12 , wherein the copolymer-linked dsDNA comprises a crosslinked copolymer.
17. The method of claim 1 , wherein the copolymer-linked dsDNA comprises at least 80% of the acrylamide-labeled dsDNA from the solution.
18. The method of claim 1 , further comprising incubating the solution of (a) for 1-3 hours.
19. The method of claim 1 , wherein the dehybridizing of (b) comprises combining the copolymer-linked dsDNA with an aqueous buffer comprising (i) sodium hydroxide and ethylenediaminetetraacetic acid (EDTA); (ii) formamide and EDTA; or (iii) Tris-EDTA.
20. The method of claim 16 , wherein the crosslinked copolymer has a molecular weight of at least 1000 kDa.
21. The method of claim 16 , wherein the separating comprises passing the solution of (b) over a microfiltration membrane to remove the crosslinked DNA-containing copolymer.
22. The method of claim 16 , wherein the separating comprises subjecting the solution of (b) to centrifugation to remove the crosslinked DNA-containing copolymer.