US 10,870,872 B2Granted

Enzymatic nucleic acid synthesis

AssigneePresident and Fellows of Harvard College

InventorsHenry Hung-yi Lee, George M. Church, Reza Kalhor

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Abstract

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.

Claims14

Dependent claims

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.

Dependencies are inferred from the claim text.

Facts

Application: 16/090,640Filed 30 Mar 2017 · us provisional application
Priority: 4 Apr 2016
Published: US 2019/0112626 A118 Apr 2019
Granted: 22 Dec 2020
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12P 19/34
Nucleotides: polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12Q 1/6844
Involving nucleic acids: nucleic acid amplification reactions
C12P
Fermentation or enzyme-using processes to synthesise a desired chemical compound or composition
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms

Family

Application62/317,919Us provisional application · filed 4 Apr 2016
US 2019/0112626 A1Pre-grant publication · published 18 Apr 2019
US 10,870,872 B2This patent

From the US filing history. Foreign family members aren’t listed.

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