Methods and compositions for DNA manipulation
View Patent ↗Methods and compositions are provided for generating a single-stranded extension on a polynucleotide molecule, the single-stranded extension having a desired length and sequence composition. Methods for forming single-stranded extensions include: the use of a cassette containing at least one nicking site and at least one restriction site at a predetermined distance from each other and in a predetermined orientation; or primer-dependent amplification which introduces into a polynucleotide molecule, a modified nucleotide which is excised to create a nick using a nicking agent. The methods and compositions provided can be used to manipulate a DNA sequence including introducing site specific mutations into a polynucleotide molecule and for cloning any polynucleotide molecule or set of joined polynucleotide molecules in a recipient molecule such as a vector of choice.
1. A nicking agent, comprising: an artificial mixture of two or more enzymes in the absence of a polynucleotide containing a modified nucleotide, wherein at least one of the enzymes is a DNA glycosylase and at least one of the enzymes is a cleaving enzyme; and wherein in the presence of the polynucleotide containing the modified nucleotide, the mixture cleaves at the modified nucleotide;
the DNA glycosylase and the cleaving enzyme having an activity ratio of at least 2:1, the activity ratio being the ratio of a first rate of forming an abasic site by excision of the modified nucleotide from the polynucleotide by DNA glycosylase to a second rate of cleaving at the abasic site in the polynucleotide by the cleaving enzyme, the first and second rates determined for a single predetermined molar amount of the modified nucleotide in the polynucleotide.
2. A nicking agent according to claim 1 , wherein the cleaving enzyme generates a 5′ phosphate in the polynucleotide molecule after excision of the modified nucleotide.
3. A nicking agent according to claim 1 , wherein the cleaving enzyme is selected from a FPG glycosylase/AP lyase and a EndoVIII glycosylase/AP lyase.
4. A nicking agent according to claim 1 , wherein the at least one cleaving enzyme generates a 3′ OH in the polynucleotide molecule after excision of the modified nucleotide.
5. A nicking agent according to claim 4 , wherein the cleaving enzyme is EndoIV endonuclease.
6. A nicking agent according to claim 1 , wherein the two or more enzymes generate a 5′ phosphate and a 3′ OH in the polynucleotide molecule after excision of the modified nucleotide.
7. A nicking agent according to claim 6 , wherein the two or more enzymes are selected from; (i) EndoIV endonuclease and EndoVIII glycosylase/AP lyase, and (ii) EndoIV endonuclease and FPG glycosylase/AP lyase.
8. A nicking agent according to claim 1 , wherein the modified nucleotide is a deoxyuridine (U).
9. A nicking agent according to claim 1 , wherein the modified nucleotide is an 8-oxo-guanine.
10. A nicking agent according to claim 1 , wherein the modified nucleotide is a deoxyinosine.
11. A nicking agent according to claims 4 or 8 , wherein the two or more enzymes are UDG glycosylase and EndoIV endonuclease.
12. A nicking agent according to claims 2 , 8 or 9 , wherein the two or more enzymes are UDG glycosylase and FPG glycosylase/AP lyase.
13. A nicking agent according to claims 4 or 10 , wherein the two or more enzymes are AlkA glycosylase and EndoIV endonuclease.
14. A nicking agent according to claims 2 or 8 , wherein the two or more enzymes are UDG glycosylase and EndoVIII glycosylase/AP lyase.
15. A nicking agent according to claims 6 or 8 , wherein the two or more enzymes are UDG glycosylase, EndoIV endonuclease and EndoVIII glycosylase/AP lyase.
16. A nicking agent according to claims 6 , 8 or 9 , wherein the two or more enzymes are UDG glycosylase, EndoIV endonuclease and FPG glycosylase/AP lyase.
17. A nicking agent according to claims 2 or 10 , wherein the two or more enzymes are AlkA glycosylase and EndoVIII glycosylase/AP lyase.
18. A nicking agent according to claims 2 or 10 , wherein the two or more enzymes are AlkA glycosylase and FPG glycosylase/AP lyase.
19. A kit, comprising a nicking agent according to claim 1 and a linearized vector.
20. A kit according to claim 19 , further comprising a DNA polymerase with 3′ to 5′ exonuclease activity.
21. A kit according to claim 19 , further comprising a T4 DNA ligase.
22. A kit according to claim 19 , further comprising at least one sequencing primer.