Methods for high fidelity production of long nucleic acid molecules

Patent metadata

Field	Value
Publication number	US-11268115-B2
Application number	US-201615359560-A
Country	US
Kind code	B2
Filing date	Nov 22, 2016
Priority date	Dec 10, 2002
Publication date	Mar 8, 2022
Grant date	Mar 8, 2022

Abstract

In a method for generating an elongated nucleic acid molecule, a nucleic acid addition of a first nucleic acid molecule attached to a first 3′ or 5′ protecting group to a nucleic acid immobilized on a surface produces an intermediate-length immobilized nucleic acid. The first protecting group is dissociated from the first nucleic acid molecule. A second nucleic acid molecule that is attached to a second associated a 3′ or 5′ associated protecting group is added to the intermediate-length nucleic acid. The second associated protecting group is dissociated from the second nucleic acid molecule. A sequentially-extended elongated immobilized nucleic acid molecule having a desired sequence and length is produced by sequentially extending the intermediate-length immobilized nucleic acid by adding additional nucleic acid molecules with associated protecting groups to the intermediate-length nucleic acid and dissociating the associated protecting group after each addition.

First claim

What is claimed is:

1. A method for generating an elongated nucleic acid molecule, the method comprising:
a) providing a nucleic acid immobilized on a surface;
b) providing a pool of nucleic acid molecules, wherein each of the nucleic acid molecules is attached to an associated 3′ or 5′ protecting group;
c) creating an intermediate-length immobilized nucleic acid comprising the nucleic acid immobilized on the surface and a first nucleic acid molecule attached to a first associated 3′ or 5′ protecting group from the pool of nucleic acid molecules by performing a nucleic acid addition of said first nucleic acid molecule to said nucleic acid immobilized on the surface;
d) after step c), dissociating said first associated 3′ or 5′ protecting group from said first nucleic acid molecule of said intermediate-length immobilized nucleic acid by applying one or more biological enzyme to the bond between said first associated 3′ or 5′ protecting group and said first nucleic acid molecule in said intermediate-length immobilized nucleic acid, thereby producing an intermediate-length immobilized nucleic acid without said first associated 3′ or 5′ protecting group;
e) creating an elongated intermediate-length immobilized nucleic acid by performing a nucleic acid addition to add a second nucleic acid molecule attached to a second associated 3′ or 5′ protecting group from the pool of nucleic acid molecules to the intermediate-length immobilized nucleic acid without said first associated 3′ or 5′ protecting group;
f) after step e), dissociating said second associated 3′ or 5′ protecting group from said second nucleic acid molecule of said elongated intermediate-length immobilized nucleic acid by applying one or more biological enzyme to the bond between said second associated 3′ or 5′ protecting group and said second nucleic acid molecule in said elongated intermediate-length immobilized nucleic acid, thereby producing an elongated intermediate-length immobilized nucleic acid without said second associated 3′ or 5′ protecting group; and
g) producing the elongated nucleic acid molecule by performing steps e) and f) for one or more times using a nucleic acid molecule attached to an associated 3′ or 5′ protecting group from the pool of nucleic acid molecules.

2. The method of claim 1 , wherein said protecting group comprises one or more protein, carbohydrate, diphosphate, phosphate derivative, nucleotide, oligonucleotide, or combinations thereof.

3. The method of claim 1 , wherein said protecting group comprises one or more protein, diphosphate, phosphate derivative, nucleotide, oligonucleotide, or combinations thereof and said one or more biological enzyme is selected from the group consisting of proteases, phosphatases, restriction enzymes, and combinations thereof.

4. The method of claim 1 , wherein the surface is a bead or particle and wherein: step c) further comprises determining whether the first nucleic acid molecule has been added to the nucleic acid immobilized on the surface of step a) by detecting whether there is an increase in electrophoretic force applied to the surface when an electric field and a magnetic field gradient are applied to the surface, wherein the increase in electrophoretic force is caused by adding the first nucleic acid molecule to the nucleic acid of step a); and step e) further comprises determining whether the second nucleic acid molecule has been added to the intermediate-length immobilized nucleic acid without said first associated 3′ or 5′ protecting group by detecting whether there is an increase in electrophoretic force applied to the surface when an electric field and a magnetic field gradient are applied to the surface, wherein the increase in electrophoretic force is caused by adding the second nucleic acid molecule to the intermediate-length immobilized nucleic acid without said first associated 3′ or 5′ protecting group.

5. A method for generating an elongated nucleic acid molecule, the method comprising:
a) providing a nucleic acid immobilized on a surface, wherein the surface is a bead or particle;
b) providing a pool of nucleic acid molecules, wherein each of the nucleic acid molecules is attached to an associated 3′ or 5′ protecting group;
c) creating an intermediate-length immobilized nucleic acid comprising the nucleic acid immobilized on the surface and a first nucleic acid molecule attached to a first associated 3′ or 5′ protecting group from the pool of nucleic acid molecules by performing a nucleic acid addition of said first nucleic acid molecule to said nucleic acid immobilized on the surface and then determining whether the first nucleic acid molecule has been added to the nucleic acid immobilized on a surface of step a) by detecting whether there is an increase in electrophoretic force applied to the surface when an electric field and a magnetic field gradient are applied to the surface, wherein the increase in electrophoretic force is caused by adding the first nucleic acid molecule to the nucleic acid of step a);
d) after step c), dissociating said first associated 3′ or 5′ protecting group from said first nucleic acid molecule of said intermediate-length immobilized nucleic acid, thereby producing an intermediate-length immobilized nucleic acid without said first associated 3′ or 5′ protecting group;
e) creating an elongated intermediate-length immobilized nucleic acid by performing a nucleic acid addition to add a second nucleic acid molecule attached to a second associated 3′ or 5′ protecting group from the pool of nucleic acid molecules to the intermediate-length immobilized nucleic acid without said first associated 3′ or 5′ protecting group and then determining whether the second nucleic acid molecule has been added to the intermediate-length immobilized nucleic acid without said first associated 3′ or 5′ protecting group by detecting whether there is an increase in electrophoretic force applied to the surface when an electric field and a magnetic field gradient are applied to the surface, wherein the increase in electrophoretic force is caused by adding the second nucleic acid molecule to the intermediate-length immobilized nucleic acid without said first associated 3′ or 5′ protecting group;
f) after step e), dissociating said second associated 3′ or 5′ protecting group from said second nucleic acid molecule of said elongated intermediate-length immobilized nucleic acid, thereby producing an elongated intermediate-length immobilized nucleic acid without said second associated 3′ or 5′ protecting group; and
g) producing the elongated nucleic acid molecule by performing steps e) and f) for one or more times using a nucleic acid molecule attached to an associated 3′ or 5′ protecting group from the pool of nucleic acid molecules.

6. The method of claim 5 , wherein said protecting group comprises one or more protein, carbohydrate, diphosphate, phosphate derivative, nucleotide, oligonucleotide, or combinations thereof.

7. The method of claim 5 , wherein said protecting group comprises one or more protein, diphosphate, phosphate derivative, nucleotide, oligonucleotide, or combinations thereof and step (d) or step (f) is accomplished by the application of one or more biological enzyme selected from the group consisting of proteases, phosphatases, restriction enzymes, and combinations thereof to the bond between said first associated 3′ or 5′ protecting group and said first nucleic acid molecule in said intermediate-length immobilized nucleic acid or to the bond between said second associated 3′ or 5′ protecting group and said second nucleic acid molecule in said elongated intermediate-length immobilized nucleic acid.

Assignees

• Massachusetts Inst Technology

Inventors

• Carr Peter A
• Chow Brian Y
• Jacobson Joseph M
• Mosley David W
• Emig Christopher

Classifications

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12Q1/6825

  Nucleic acid detection involving sensors · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q2565/518

  characterised by the immobilisation of the nucleic acid sample or target · CPC title