Methods for high fidelity production of long nucleic acid molecules

• US 9,499,848 B2
• Filed: 08/13/2013
• Issued: 11/22/2016
• Est. Priority Date: 12/10/2002
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Carr, Peter A., Chow, Brian Y., Jacobson, Joseph M., Mosley, David W., Emig, Christopher
• Primary Examiner(s): Lu, Frank
• Application Number:
  US13/966,238
• Publication Number:
  US 20130323722A1
• Time in Patent Office: 1,197 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/283.1, 435/287.1, 435/287.2, 436/94, 436/501, 536/23.1, 536/24, 536/3, 536/24.33, 536/25.3
• US Class Current: 1/1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 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

Abstract

In a method for generating a long nucleic acid molecule, nucleic acids immobilized on a surface and having overlapping complementary sequences is released into solution. The overlapping complementary sequences are hybridized to form hybridized nucleic acids, followed by extension or ligation of the hybridized nucleic acids to synthesize the long nucleic acid molecule. The nucleic acids may comprise first and second series of nucleic acids having redundant overlapping sequences, wherein nucleic acids from the first and second series are complementary to each other. The complementary nucleic acids are hybridized to form the hybridized nucleic acids. The generated long nucleic acid molecule may have a predetermined sequence element, and it may be introduced into a system wherein the predetermined sequence element is required for replication, such that replication of the synthesized long nucleic acid molecule is indicative of the presence of the predetermined sequence element in the long nucleic acid molecule.

Claims

1. A method for generating a long nucleic acid molecule, the method comprising the steps of:

a) providing a plurality of nucleic acids immobilized on a surface, wherein said plurality of nucleic acids comprises nucleic acids having overlapping complementary sequences, wherein said overlapping complementary sequences are capable of hybridizing to each other;

b) releasing said plurality of nucleic acids into a solution; and

c) producing the long nucleic acid molecule by the steps of;

i) forming a plurality of hybridized nucleic acids by hybridizing said overlapping complementary sequences to each other;

ii) synthesizing a plurality of intermediate-length nucleic acid duplexes by extending said plurality of hybridized nucleic acids;

iii) forming intermediate-length nucleic acids having overlapping complementary sequences by disassociating said plurality of intermediate-length nucleic acid duplexes, wherein the overlapping complementary sequences in said intermediate-length nucleic acids are capable of hybridizing to each other;

iv) forming a plurality of intermediate-length hybridized nucleic acids by hybridizing the intermediate-length nucleic acids having overlapping complementary sequences to each other;

v) synthesizing a plurality of extended intermediate-length nucleic acid duplexes by extending said plurality of intermediate-length hybridized nucleic acids;

vi) generating a plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule by repeating steps iii) to v) for multiple cycles until said plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule are produced, wherein, in each of the cycles, the plurality of intermediate-length nucleic acid duplexes in step iii) is replaced with the plurality of extended intermediate-length nucleic acid duplexes produced in step v); and

vii) forming the long nucleic acid molecule by ligating the plurality of nucleic acid duplexes having the sequence of the long nucleic acid molecule and covering full length of the long nucleic acid molecule.

2. The method of claim 1, wherein the nucleic acids immobilized on the surface are synthesized on the surface.

3. The method of claim 1, wherein the nucleic acids immobilized on the surface are synthesized separately and then attached to the surface.

4. The method of claim 1, wherein each of the plurality of nucleic acids immobilized on the surface is synthesized by the steps of:

d) providing a first immobilized nucleic acid comprising a first 5′

region and a first 3′

region;

e) providing a second immobilized nucleic acid comprising a second 5′

region and a second 3′

region, wherein said second 3′

region and said first 5′

region comprise identical nucleic acid sequences;

f) hybridizing said first immobilized nucleic acid with an oligonucleotide under conditions promoting hybridization of said oligonucleotide to said first 3′

region, extending the oligonucleotide hybridized to said first 3′

region, and producing a first extension product comprising a 3′

region that is complementary to said first 5′

region; and

g) hybridizing said second immobilized nucleic acid with said first extension product under conditions promoting hybridization of said 3′

region of said first extension product to said second 3′

region, extending the 3′

region of said first extension product, and producing a second extension product comprising a 3′

region that is complementary to said second 5′

region, wherein said second extension product comprises a sequence complementary to said first 3′ and

5′

regions and said second 3′ and

5′

regions and said second extension product comprises the nucleic acids immobilized on the surface.

5. The method of claim 1, wherein the long nucleic acid molecule is deoxyribonucleic acid or ribonucleic acid.

6. The method of claim 1, wherein a ligase or polymerase is employed in the method to synthesize the long nucleic acid molecule.

7. The method of claim 1, further comprising the step of determining whether the long nucleic acid molecule is error-free.

8. The method of claim 1, wherein the long nucleic acid molecule comprises a predetermined sequence element and further comprising the steps of:

h) introducing said long nucleic acid molecule into a system wherein said predetermined sequence element is required for replication and wherein replication of said long nucleic acid molecule in the system is indicative of the presence of said predetermined sequence element in said long nucleic acid molecule;

i) providing conditions promoting replication of said long nucleic acid molecule; and

j) detecting the replication of said long nucleic acid molecule within said system.

9. The method of claim 1, wherein said nucleic acids having overlapping complementary sequences comprise a first series of nucleic acids having redundant overlapping sequences and a second series of nucleic acids having redundant overlapping sequences, and wherein nucleic acids from said first series of nucleic acids are complementary to nucleic acids from said second series of nucleic acids.

10. The method of claim 9, wherein each of the plurality of nucleic acids is synthesized by the steps of:

d) providing a first immobilized nucleic acid comprising a first 5′

region and a first 3′

region;

e) providing a second immobilized nucleic acid comprising a second 5′

region and a second 3′

region, wherein said second 3′

region and said first 5′

region comprise identical nucleic acid sequences;

f) hybridizing said first immobilized nucleic acid with an oligonucleotide under conditions promoting hybridization of said oligonucleotide to said first 3′

region, extending the oligonucleotide hybridized to said first 3′

region, and producing a first extension product comprising a 3′

region that is complementary to said first 5′

region; and

g) hybridizing said second immobilized nucleic acid with said first extension product under conditions promoting hybridization of said 3′

region of said first extension product to said second 3′

region, extending the 3′

region of said first extension product, and producing a second extension product comprising a 3′

region that is complementary to said second 5′

region, wherein said second extension product comprises a sequence complementary to said first 3′ and

5′

regions and said second 3′ and

5′

regions and said second extension product comprises the nucleic acids immobilized on the surface.

11. The method of claim 9, wherein the long nucleic acid molecule is deoxyribonucleic acid or ribonucleic acid.

12. The method of claim 9, wherein a ligase or polymerase is employed in the method to synthesize the long nucleic acid molecule.

13. The method of claim 9, further comprising the step of determining whether the synthesized long nucleic acid molecule is error-free.

14. The method of claim 9, wherein the long nucleic acid molecule comprises a predetermined sequence element and further comprising the steps of:

h) introducing said synthesized long nucleic acid molecule into a system wherein said predetermined sequence element is required for replication and wherein replication of said synthesized long nucleic acid molecule in the system is indicative of the presence of said predetermined sequence element in said synthesized long nucleic acid molecule;

i) providing conditions promoting replication of said synthesized long nucleic acid molecule; and

j) detecting the replication of said synthesized long nucleic acid molecule within said system.