Products and methods for reducing dye artifacts
View Patent ↗The present invention provides products and methods of reducing dye artifacts from chain extension reactions.
1. A method of reducing dye artifacts from a polynucleotide chain extension reaction, comprising: a) contacting a polynucleotide chain extension reaction solution that comprises protein with at least one protein binding material to form a complex of the protein binding material with the protein; and b) separating the complex from the chain extension reaction solution.
2. The method of claim 1 , wherein the protein is an enzyme.
3. The method of claim 2 , wherein the enzyme is a DNA polymerase or RNA polymerase.
4. The method of claim 3 , wherein the DNA polymerase is a thermostable DNA polymerase.
5. The method of claim 3 , wherein the DNA polymerase is a Taq DNA polymerase.
6. The method of claim 5 , wherein the mutated Taq DNA polymerase comprises a F667Y mutation.
7. The method of claim 1 , wherein the protein is associated with a dye-labeled dideoxynucleotide.
8. The method of claim 1 , wherein the chain extension reaction is a DNA sequencing reaction.
9. The method of claim 1 , wherein the protein binding material is a membrane.
10. The method of claim 9 , wherein the membrane is a porous membrane.
11. The method of claim 10 , wherein the porous membrane comprises pores ranging from about 0.01 μm to about 200 μm in size.
12. The method of claim 11 , wherein the porous membrane comprises pores ranging from about 0.05 μm to about 100 μm in size.
13. The method of claim 12 , wherein the porous membrane comprises pores ranging from about 0.1 μm to about 50 μm in size.
14. The method of claim 10 , wherein the porous membrane has a thickness ranging from about 0.1 μm to about 2000 μm.
15. The method of claim 14 , wherein the porous membrane has a thickness ranging from about 0.5 μm to about 1500 μm.
16. The method of claim 15 , wherein the porous membrane has a thickness ranging from about 0.5 μm to about 1000 μm.
17. The method of claim 1 , wherein the protein binding material is a solid support.
18. The method of claim 17 , wherein the solid support is a microsphere.
19. The method of claim 18 , wherein the microsphere is a polystyrene microsphere.
20. The method of claim 18 , wherein the microsphere is a polymer microsphere having a diameter from about 100 nm to about 2000 μm.
21. The method of claim 20 , wherein the microsphere has a diameter from about 500 nm to about 1000 μm.
22. The method of claim 20 , wherein the microsphere has a diameter from about 1 μm to about 200 μm.
23. The method of claim 18 , wherein the microsphere is a nonporous microsphere.
24. The method of claim 18 , wherein the microsphere is a microporous microsphere.
25. The method of claim 18 , wherein the microsphere is a macroporous microsphere.
26. The method of claim 18 , wherein the microsphere has sores with a size greater than about 4000 Angstroms.
27. The method of claim 1 , wherein the protein binding material forms a layer on the surface of a solid support.
28. The method of claim 27 , wherein the solid support is a porous membrane.
29. The method of claim 27 , wherein the protein binding material is associated with the solid support via covalent bonds.
30. The method of claim 1 , wherein the protein binding material is mixed with a size-based purification media.
31. The method of claim 1 , wherein the protein binding material is layered on a size-exclusion spin column.
32. The method of claim 1 , further comprising performing cycle sequencing prior to step (a).
33. The method of claim 1 , further comprising purifying the chain extension reaction solution.
34. The method of claim 33 , wherein the chain extension reaction solution is purified by size-based purification.
35. The method of claim 34 , wherein the chain extension reaction solution is purified by size-based purification using a size-exclusion spin column.
36. The method of claim 34 , wherein the chain extension reaction solution is purified by size-based purification using size-excluded ion exchange.
37. The method of claim 1 , further comprising analyzing the chain extension reaction solution on a fluorescent DNA sequencer.
38. The method of claim 33 , wherein the chain extension reaction solution is contacted with the protein binding material during the purification step.
39. The method of claim 33 , wherein the chain extension reaction solution is contacted with the protein binding material prior to the purification step.
40. The method of claim 1 , wherein the protein binding material is contacted with the chain extension reaction solution prior to chain extension reaction.
41. The method of claim 40 , wherein the protein binding material remains in contact with the chain extension reaction solution during chain extension reaction.