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 for producing a synthon, comprising:
(a) making a reaction mix comprising:
(i) a polymerase comprising:
an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:3;
an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:1; and
an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2;
(ii) an exonuclease;
(iii) a ligase;
(iv) dNTPs; and
(v) a set of overlapping polynucleotides; and
(b) incubating the reaction mix under suitable reaction conditions to produce a synthon that contains sequence from each of the overlapping polynucleotides.
2. The method of claim 1 , wherein step (a) is done by adding a composition comprising the polymerase, exonuclease, ligase and dNTPs to a composition comprising the set of overlapping polynucleotides.
3. The method of claim 1 , wherein step (b) comprises incubating the reaction mix at temperature of at least 50° .
4. The method of claim 1 , wherein step (b) is an isothermal incubation.
5. The method of claim 1 , wherein step (b) comprises thermocycling.
6. The method of claim 1 , further comprising introducing the synthon of step (b) into a cell by transformation.
7. The method of claim 1 , further comprising amplifying the synthon of step (b) by polymerase chain reaction (PCR).
8. The method of claim 1 , wherein each of the overlapping polynucleotides is at a concentration in the range of 0.02 nM to 100 nM.
9. The method of claim 1 , wherein at least two of the oligonucleotides in the set have an overlap of at least 15 nucleotides.
10. The method of claim 1 , wherein the set of overlapping polynucleotides has at least two members.
11. The method of claim 1 , wherein the set of overlapping polynucleotides has at least five members.
12. The method of claim 1 , wherein the overlapping polynucleotides each has a length in the range of 100 bases to 30 kb.
13. The method of claim 1 , wherein the overlapping polynucleotides each has a length of at least 2 kb.
14. The method of claim 1 , wherein the set of overlapping polynucleotides overlapping polynucleotides comprises:
double-stranded polynucleotides;
at least one double strand polynucleotide and at least one single strand oligonucleotide;
single stranded oligonucleotides; and/or
a subpopulation of polynucleotides that are otherwise identical to one another except for a sequence that varies between the members of the sub-population.
15. The method of claim 1 , wherein the polymerase comprises:
(i) an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:3,
(ii) an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:1; and
(iii) an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:2.
16. The method of claim 1 , wherein the polymerase comprises the amino acid sequence of SEQ ID NO:3.
17. The method of claim 1 , wherein the exonuclease is a 5′−3′ exonuclease.
18. The method of claim 1 , wherein the ligase is thermostable.
19. The method of claim 1 , wherein the reaction mix further comprises a single strand DNA binding protein.
20. The method of claim 1 , wherein the reaction mix does not comprise a crowding agent.
21. The method of claim 1 , wherein the reaction mix does not comprise a non strand-displacing polymerase.
22. The method of claim 1 , wherein the reaction mix comprises KCI at a concentration in the range of 7 mM to 150 mM.