Fusion polymerase and method for using the same
This disclosure provides, among other things, a composition comprising: comprising a fusion protein comprising: (a) a DNA polymerase; and (b) a heterologous sequence-specific DNA binding domain. A method for copying a DNA template, as well as a kit for performing the same, are also described.
1. A method comprising;
(a) producing a reaction mixture comprising:
(i) a fusion protein comprising a strand-displacing polymerase and a sequence specific DNA binding domain;
(ii) a plurality of polynucleotides having overlapping sequences; and
(iii) dNTPs; and
(b) incubating the reaction mixture under suitable reaction conditions to produce a synthon that contains sequence from each of the plurality of polynucleotides.
2. The method of claim 1 , wherein the reaction mix in (a) further comprises a 5′ to 3′ exonuclease for producing in (b), complementary single strand overhangs in the plurality of polynucleotides having overlapping sequences.
3. The method of claim 2 , wherein step (b) further comprises hybridizing the complementary overhangs in the plurality of polynucleotides and filling in any single stranded gaps by the strand-displacing polymerase.
4. The method of claim 3 , wherein the reaction mix further comprises a ligase, for sealing nicks after the gaps are filled.
5. The method of claim 3 , wherein the 5′ to 3′ exonuclease removes any DNA flaps that are produced by the strand displacing polymerase.
6. The method according to claim 3 , wherein the reaction mixture further comprises T5 exonuclease at a concentration range of 0.0004 U/μl-0.064 U/μl.
7. The method of claim 1 , wherein the method further comprises cloning the synthon into a vector.
8. The method of claim 7 , wherein the vector has a selectable marker.
9. The method according to claim 8 , further comprising transforming cells with the vector wherein the selectable marker is lacZ and wherein any transformed cells that comprise the synthon are white while the cells that do not contain the synthon are blue.
10. The method according to claim 1 , further comprising sequencing the synthon to produce synthon sequence data.
11. The method according to claim 10 further comprising comparing the synthon sequence data to the sequences of the plurality of polynucleotides.
12. The method according to claim 1 , wherein the reaction mixture further comprises a single stranded binding protein and/or a crowding agent.
13. The method according to claim 1 , wherein in (a) the plurality of polynucleotides are at a concentration in the range of 0.02 nM-100 nM.
14. The method according to claim 1 , wherein the plurality of polynucleotides are overlapping amplicons, restriction endonuclease cleaved fragments or synthetic oligonucleotides.