Enzymatic nucleic acid synthesis
The disclosure provides methods for making a polynucleotide wherein the addition of nucleotides can be physically, chemically and/or enzymatically controlled. The methods include combining a selected nucleotide, cations, an error prone or template independent DNA polymerase at a reaction site including an initiator sequence attached thereto and having a 3′ terminal nucleotide, wherein the reaction reagents can be modulated and under conditions that allow covalent addition of one or more of a selected nucleotide to the 3′ terminal nucleotide such that the selected nucleotide becomes a 3′ terminal nucleotide, and repeating the addition step until the polynucleotide is formed.
1. A method for making a polynucleotide comprising
(a) combining a selected nucleotide triphosphate, cations, a template independent DNA polymerase, and a nucleotide triphosphate inactivating enzyme at a reaction site on a support surface including an initiator sequence attached thereto and having a 3′ terminal nucleotide, wherein reaction reagents are present at selected concentrations and under conditions which covalently add one or more of the selected nucleotide triphosphate to the 3′ terminal nucleotide such that the selected nucleotide triphosphate becomes a 3′ terminal nucleotide and under conditions which inactivate free nucleotide triphosphates until free nucleotide triphosphates are substantially inactivated, wherein a desired number of the selected nucleotide triphosphate is added to the initiator sequence, and
(b) repeating step (a) until the polynucleotide is formed.
2. The method of claim 1 wherein the nucleotide triphosphate inactivating enzyme is a nucleotide triphosphate degrading enzyme.
3. The method of claim 1 wherein the nucleotide triphosphate inactivating enzyme is a nucleotide triphosphate degrading enzyme that degrades nucleotide triphosphates at a rate slower than rate of addition of nucleotides by the template independent DNA polymerase.
4. The method of claim 1 wherein the nucleotide triphosphate inactivating enzyme is a nucleotide triphosphate degrading enzyme present at a concentration that degrades nucleotide triphosphates at a rate slower than rate of addition of nucleotides by the present concentration of the template independent DNA polymerase.
5. The method of claim 1 wherein the nucleotide triphosphate inactivating enzyme comprises ATP diphosphohydrolase, dNTP pyrophosphatases, dNTPases, and phosphatases.
6. The method of claim 1 wherein the concentration of nucleotide triphosphate inactivating enzyme is modulated to control addition of one or more nucleotides.
7. The method of claim 1 wherein the nucleotide triphosphate inactivating enzyme renders free nucleotide triphosphates inactive by degradation.
8. The method of claim 1 wherein the nucleotide triphosphate inactivating enzyme renders free nucleotide triphosphates inactive by polymerizing them with each other.
9. The method of claim 1 wherein the selected nucleotide triphosphate is added to the reaction site on the support surface including the initiator sequence having the terminal nucleotide, the template independent DNA polymerase and the nucleotide triphosphate inactivating enzyme.
10. The method of claim 1 wherein the template independent DNA polymerase and the nucleotide triphosphate inactivating enzyme are added to the reaction site on the support surface including the initiator sequence having the terminal nucleotide, and the selected nucleotide triphosphate.
11. The method of claim 10 wherein the reaction reagents are removed from the reaction site on the support surface and additional reaction reagents are provided to the reaction site on the support surface after each round of addition.
12. The method of claim 1 wherein the nucleotide triphosphate inactivating enzyme is added to the reaction site on the support surface including the initiator sequence having the terminal nucleotide, the template independent DNA polymerase and the selected nucleotide triphosphate under conditions where the polymerase is inactive, and wherein the polymerase is activated upon addition of the nucleotide triphosphate inactivating enzyme.
13. The method of claim 12 wherein the reaction reagents are removed from the reaction site on the support surface and additional reaction reagents are provided to the reaction site on the support surface after each round of addition.
14. The method of claim 1 wherein step (b) is repeated a plurality of times after which the reaction reagents are removed from the reaction site on the support surface and additional reaction reagents are provided to the reaction site on the support surface.