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

• US 7,932,025 B2
• Filed: 12/10/2003
• Issued: 04/26/2011
• Est. Priority Date: 12/10/2002
• Status: Expired due to Fees

• 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.
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/733,855
• Publication Number:
  US 20050227235A1
• Time in Patent Office: 2,694 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6.11
• 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 determining whether a nucleotide or an oligonucleotide is added to a nucleic acid, the method comprising:

a) providing a solid support having an immobilized nucleic acid;

b) performing a nucleic acid addition reaction by adding a nucleotide or an oligonucleotide to the nucleic acid immobilized on the solid support to synthesize a nucleic acid molecule on the solid support;

c) after the nucleic acid addition reaction, passing the solid support through a channel opening with at least one detector, wherein the at least one detector detects the length of a nucleic acid;

d) determining whether the nucleotide or the oligonucleotide is added to the nucleic acid immobilized on the solid support using the at least one detector, wherein an increase in length of the nucleic acid molecule on the solid support indicates that the nucleotide or the oligonucleotide is added to the nucleic acid immobilized on the solid support; and

e) repeating steps b) through d) if the nucleotide or the oligonucleotide is not added to the nucleic acid immobilized on the solid support.

2. The method of claim 1, wherein the solid support is a bead or particle and further comprising the step of detecting whether there is an increase in electrophoretic force applied to the solid support when an electric field and a magnetic field gradient are applied to the solid support, wherein the increase in electrophoretic force applied to the solid support is caused by adding the nucleotide or the oligonucleotide to the nucleic acid immobilized on the solid support.

3. A method for determining whether a nucleotide or an oligonucleotide is added to a nucleic acid, the method comprising:

a) providing a solid support having an immobilized nucleic acid;

b) performing a nucleic acid addition reaction by adding a nucleotide having a fluorescent 5′

protecting group or an oligonucleotide having a fluorescent 5′

protecting group to the nucleic acid immobilized on the solid support to synthesize a nucleic acid molecule having a fluorescent 5′

protecting group on the solid support;

c) after the nucleic acid addition reaction, passing the solid support through a channel opening with at least one detector, wherein the at least one detector detects fluorescent signals;

d) determining whether the nucleotide having a fluorescent 5′

protecting group or the oligonucleotide having a fluorescent 5′

protecting group is added to the nucleic acid immobilized on the solid support using the at least one detector, wherein the presence of the nucleic acid molecule having a fluorescent 5′

protecting group on the solid support indicates that the nucleotide having a fluorescent 5′

protecting group or the oligonucleotide having a fluorescent 5′

protecting group is added to the nucleic acid immobilized on the solid support; and

e) repeating steps b) through d) if the nucleotide having a fluorescent 5′

protecting group or the oligonucleotide having a fluorescent 5′

protecting group is not added to the nucleic acid immobilized on the solid support.

4. The method of claim 3, wherein the solid support is a bead or particle and further comprising the step of detecting whether there is an increase in electrophoretic force applied to the solid support when an electric field and an magnetic field gradient are applied to the solid support, wherein the increase in electrophoretic force applied to the solid support is caused by adding the nucleotide or the oligonucleotide to the nucleic acid immobilized on the solid support.

5. The method of claim 3, further comprising the steps of:

if step d) indicates the presence of the nucleic acid molecule having a fluorescent 5′

protecting group on the solid support, deprotecting the nucleic acid molecule by washing the nucleic acid molecule using a deprotection wash to remove the fluorescent 5′

protecting group of the nucleic acid molecule, and producing an after-washing solution;

flowing the after-washing solution through a channel opening;

monitoring the after-washing solution through the channel opening for the presence of a removed 5′

protecting group; and

repeating the deprotecting, flowing, and monitoring steps if the removed 5′

protecting group is not detected in the after-washing solution through the channel opening or adding a nucleotide having a 5′

protecting group or an oligonucleotide having a 5′

protecting group to the nucleic acid molecule on the solid support if the removed 5′

protecting group is detected in the after-washing solution through the channel opening, thereby preventing errors in the sequence of the nucleic acid molecule.

6. The method of claim 5, wherein the solid support is a bead or particle and further comprising the step of detecting whether there is an increase in electrophoretic force applied to the solid support when an electric field and an magnetic field gradient are applied to the solid support, wherein the increase in electrophoretic force applied to the solid support is caused by adding the nucleotide or the oligonucleotide to the nucleic acid immobilized on the solid support.

7. The method of claim 5, wherein the errors in the sequence of the nucleic acid molecule are selected from the group consisting of insertion errors, deletion errors, and wrong base incorporation errors.

8. The method of claim 5, wherein said monitoring step is carried out using a plurality of light sources and fluorescence detectors.

9. A method for preventing errors in a nucleic acid molecule, the method comprising:

a) synthesizing a nucleic acid molecule having a 5′

protecting group by adding a nucleotide having a 5′

protecting group or an oligonucleotide having a 5′

protecting group to a nucleic acid;

b) deprotecting the nucleic acid molecule by washing the nucleic acid molecule using a deprotection wash to remove the 5′

protecting group of the nucleic acid molecule, and producing an after-washing solution;

c) flowing the after-washing solution through a channel opening and monitoring the after-washing solution through the channel opening for the presence of a removed 5′

protecting group; and

d) repeating steps b) and c) if the removed 5′

protecting group is not detected in the after-washing solution through the channel opening or adding a nucleotide having a 5′

protecting group or an oligonucleotide having a 5′

protecting group to the nucleic acid molecule if the removed 5′

protecting group is detected in the after-washing solution through the channel opening, thereby preventing errors in the sequence of the nucleic acid molecule.

10. The method of claim 9 wherein the errors in the sequence of the nucleic acid molecule are selected from the group consisting of insertion errors, deletion errors, and wrong base incorporation errors.

11. The method of claim 9, wherein the 5′

protecting group of the nucleic acid molecule is a fluorescent 5′

protecting group and said monitoring step is carried out using a plurality of light sources and fluorescence detectors.