Methods of elongating DNA
The present invention relates to methods of elongating chromosomes. Embodiments of the present disclosure are directed to methods of elongating DNA by immobilizing or attaching the DNA to a substrate. According to one aspect, naturally occurring DNA includes a nucleic acid and one or more factors bound thereto, and may be referred to herein as “starting DNA”.
1. A method of sequencing elongated DNA comprising
attaching DNA to a substrate by one or more covalent bonds, an antibody interaction, a ligand-ligand interaction, or a telomere-associated factor, wherein the DNA includes one or more factors bound thereto,
contacting the DNA with at least one reagent which removes the one or more factors from the DNA,
elongating the DNA as the one or more factors are removed from the DNA,
passing elongated DNA through a nanopore under an electrical potential,
measuring a change in current resulting from a nucleotide of the elongated DNA passing through the nanopore, and
identifying the nucleotide based on the change in current.
2. The method of claim 1 wherein the elongated DNA is passed through the nanopore in a back and forth direction.
3. The method of claim 1 wherein the elongated DNA is circular elongated DNA.
4. The method of claim 3 wherein the circular elongated DNA is rotated through the nanopore.
5. The method of claim 3 wherein the circular elongated DNA is continuously rotated through the nanopore.
6. The method of claim 3 wherein the circular elongated DNA is revolved through the nanopore.
7. The method of claim 3 wherein the circular elongated DNA is continuously revolved through the nanopore.
8. The method of claim 3 wherein the circular elongated DNA spins through the nanopore.
9. The method of claim 3 wherein the circular elongated DNA continuously spins through the nanopore.
10. The method of claim 1 wherein the one or more factors includes a protein, RNA, carbohydrate, lipid or nonchromosomal DNA.
11. The method of claim 1 wherein the one or more factors are one or more chromatin factors.
12. The method of claim 1 wherein the DNA is double stranded DNA, single stranded DNA, or chromosomal DNA.
13. The method of claim 1 wherein the DNA has a first end and a second end and wherein the first end of the DNA is attached to the substrate.
14. The method of claim 1 wherein the DNA is elongated under influence of a flowing reagent.
15. The method of claim 1 wherein the DNA is elongated under influence of an electric field.
16. The method of claim 1 wherein the DNA is elongated under influence of gravity.
17. The method of claim 1 wherein the DNA has a first end and a second end and wherein the DNA is attached to the substrate at the first end and the second end.
18. The method of claim 1 wherein the DNA has a first end and a second end and wherein the DNA is attached to the substrate at the first end and the second end and wherein the first end and second end are separated a distance of at least less than the length of the DNA.
19. The method of claim 1 wherein the DNA has a first end and a second end and wherein the DNA is attached to the substrate at a location intermediate the first end and the second end.
20. The method of claim 1 wherein the DNA has a first end and a second end and the first end of the DNA is attached to the substrate, wherein the at least one reagent is flowed from a first end of the DNA to a second end of the DNA.
21. The method of claim 1 wherein the DNA has a first end and a second end and the first end of the DNA is attached to the substrate, wherein the at least one reagent is flowed from a first end of the DNA to a second end of the DNA whereby factors are removed from the DNA by the at least one reagent and the DNA is elongated under the influence of the flowing reagent.
22. The method of claim 1 wherein the DNA has a first end and a second end and the first end of the DNA is attached to the substrate, wherein the at least one reagent is flowed one or more times from a first end of the DNA to a second end of the DNA.
23. The method of claim 1 wherein the DNA has a first end and a second end and the first end of the DNA is attached to the substrate, wherein the at least one reagent is flowed one or more times from a first end of the DNA to a second end of the DNA whereby factors are removed from the DNA by the at least one reagent and the DNA is elongated under the influence of the flowing reagent.
24. The method of claim 1 wherein the DNA is contacted with two or more reagents which remove the one or more factors from the DNA.
25. The method of claim 1 wherein the DNA is contacted with two or more reagents in series which remove the one or more factors from the DNA.
26. The method of claim 1 including a plurality of DNA attached to the substrate and wherein the plurality of DNA is contacted with at least one reagent which removes the one or more factors from the DNA, and wherein the plurality of DNA is elongated as the one or more factors are removed from the plurality of DNA.
27. The method of claim 1 including a plurality of DNA attached to the substrate and wherein the plurality of DNA is contacted with two or more reagents which remove the one or more factors from the plurality of DNA, and wherein the plurality of DNA is elongated as the one or more factors are removed from the plurality of DNA.
28. The method of claim 1 wherein the substrate is a planar substrate.
29. The method of claim 1 wherein the substrate is a planar substrate and wherein the at least one reagent flows across the surface of the planar substrate to remove the one or more factors from the DNA and to elongate the DNA.
30. The method of claim 1 wherein the substrate is a planar substrate, wherein the DNA has a first end and a second end and the first end of the DNA is attached to the planar substrate and wherein the at least one reagent flows across the surface of the planar substrate from the first end of the DNA to the second end of the DNA to remove the one or more factors from the DNA and to elongate the DNA.
31. The method of claim 1 wherein the substrate is a planar substrate including a plurality of DNA having a first end and a second end and being attached to the substrate at the first end and wherein the at least one reagent flows across the surface of the planar substrate from the first end of the DNA to the second end of the DNA to remove the one or more factors from the plurality of DNA and to elongate the plurality of DNA.