Methods for high fidelity production of long nucleic acid molecules with error control

• US 20050227235A1
• Filed: 12/10/2003
• Published: 10/13/2005
• Est. Priority Date: 12/10/2002
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Jacobson, Joseph M., Carr, Peter A., Emig, Christopher, Chow, Brian Y., Mosley, David W.
• Granted Patent: US 7,932,025 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

This invention generally relates to nucleic acid synthesis, in particular DNA synthesis. More particularly, the invention relates to the production of long nucleic acid molecules with precise user control over sequence content. This invention also relates to the prevention and/or removal of errors within nucleic acid molecules.

Claims

1. A method for generating a nucleic acid molecule with precise user control, the method comprising:

a) providing a plurality of nucleic acids immobilized on a surface;

b) providing a nucleic acid molecule attached to a protecting group;

c) contacting said immobilized nucleic acid molecule with said nucleic acid molecule attached to a protecting group;

d) elongating said immobilized nucleic acid molecule; and

e) dissociating said immobilized nucleic acid molecule from said protecting group thereby extending said immobilized nucleic acid molecule.

2. The method of claim 1, wherein said protecting group comprises proteins, carbohydrates;

diphosphates, phosphate derivatives, nucleotides, oligonucleotides, or combinations thereof.

3. The method of claim 1, wherein the removal of said protecting group is accomplished by the application of heat, proteases, phosphotases, restriction enzymes, or combinations thereof.

4. A method for removing or controlling errors in nucleic acid molecules comprising arbitrary user-specified sequence composition and length, the method comprising:

a) providing a solid support for synthesis of nucleic acid;

b) synthesizing nucleic acid attached to the solid support;

c) passing the solid support and newly synthesized nucleic acid through a channel opening;

d) detecting errors in nucleic acid synthesis; and

e) correcting errors in nucleic acid synthesis.

5. A method for removing or controlling errors in nucleic acid molecules comprising arbitrary user-specified sequence composition and length, the method comprising:

a) synthesizing nucleic acids with 5′

protecting groups;

b) monitoring the deprotection of the 5′

protecting group using a channel opening;

c) detecting errors in nucleic acid synthesis; and

d) correcting errors in nucleic acid synthesis.

6. A method for detecting the addition of nucleotides to nucleic acid molecules comprising:

a) providing a solid support for the attachment of a nucleic acid;

b) elongating said attached nucleic acid;

c) detecting the force exerted on the growing nucleic acid molecule;

d) detecting errors in nucleic acid synthesis; and

e) correcting errors in nucleic acid synthesis.

7. A method for removing or controlling errors in nucleic acid molecules comprising arbitrary user-specified sequence composition and length, the method comprising the parallelization of single-molecule systems with and without arrays of light sources and detectors.

8. A method for the microfabrication of quadrupole arrays, the method comprising:

a) defining bond pads and lithographically defining and depositing a layer of diagonal metal wires on a silicon substrate;

b) lithographically designing and depositing a film of soft magnetic material over a portion of the metal lines; and

c) lithographically defining and depositing a second layer of metal lines over the magnetic film layer.

9. A method for independently controlling particles, the method comprising controlling said particles via an electric field, a magnetic field, optical tweezers, or any combinations thereof.

10. A method for synthesizing polymers in which individual molecules are monitored during synthesis to observe and evaluate the reaction products.