Methods for assembly of high fidelity synthetic polynucleotides

• US 20070269870A1
• Filed: 10/18/2005
• Published: 11/22/2007
• Est. Priority Date: 10/18/2004
• Status: Abandoned Application

• Current Assignee: Codon Devices Incorporated
• Original Assignee: Codon Devices Incorporated
• Inventors: Church, George, Jacobson, Joseph, Afeyan, Noubar, Baynes, Brian, Chapman, Brad, Strack-Logue, Bettina, Nesmith, Kenneth
• Application Number:
  US11/254,250
• Publication Number:
  US 20070269870A1
• Time in Patent Office: Days
• US Class Current: 435/91.200
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation

Abstract

Disclosed are methods of manufacturing synthetic DNAs, that is, DNAs made at least in significant part by chemical synthesis of nucleic acid polymers. Also provided are methods for assembling plural DNAs in the same pool by multiplexed assembly of synthetic oligonucleotides. In exemplary embodiments, the methods involve pre-amplification of one or more oligonucleotides using “universal” primers, reduction of the error rate in oligonucleotide and/or nucleic acid products, and sequence optimization and oligonucleotides design. Also provided are low-purity arrays of nucleic acids and methods for assembling nucleic acids using oligonucleotides obtained from low-purity arrays.

Claims

1-291. -291. (canceled)

292. A method for assembling at least one polynucleotide construct having a predefined sequence, comprising:

a) providing one or more nucleic acid arrays comprising a solid support and a plurality of discrete features associated with said solid support, wherein each feature independently comprises a population of construction oligonucleotides collectively having a defined consensus sequence but in which no more than 10 percent of said construction oligonucleotides of said feature have the identical sequence;

b) simultaneously or sequentially releasing construction oligonucleotides from one or more of said features to form a pool of construction oligonucleotides comprising partially overlapping sequences that define the sequence of said polynucleotide construct; and

c) providing conditions promoting;

i) hybridization of complementary, overlapping sequences of said construction oligonucleotides;

ii) ligation, chain extension, or chain extension and ligation, of hybridized construction oligonucleotides to form a polynucleotide construct;

iii) error reduction to provide a polynucleotide construct having said predetermined sequence.

293. The method of claim 292, wherein said polynucleotide construct is at least 1000 bases in length.

294. The method of claim 292, wherein said polynucleotide construct is at least 5000 bases in length.

295. The method of claim 292, wherein said pool of construction oligonucleotides comprises positive and negative strands that are complementary in the overlapping regions.

296. The method of claim 292, further comprising amplifying said pool of construction oligonucleotides prior to forming the polynucleotide construct.

297. The method of claim 292, further comprising subjecting said pool of construction oligonucleotides to an error reduction process prior to forming the polynucleotide construct.

298. The method of claim 292, wherein said error reduction is an error filtration process.

299. The method of claim 292, wherein said error reduction is an error neutralization process.

300. The method of claim 292, wherein said error reduction is an error correction process.

301. The method of claim 292, wherein construction oligonucleotides comprise at least one pair of primer hybridization sites flanking at least a portion of said oligonucleotides and common to at least a subset of said construction oligonucleotides.

302. The method of claim 301, wherein said primer hybridization sites are removable.

303. The method of claim 292, wherein at least two polynucleotide constructs are formed in the same reaction mixture.

304. The method of claim 292, further comprising amplifying the polynucleotide construct.

305. The method of claim 292, further comprising repeating c) at least twice to provide a polynucleotide construct having said predetermined sequence.

306. The method of claim 292, wherein said error reduction utilizes a single stranded nuclease.

307. The method of claim 306, further comprising a round of denaturation and renaturation prior to error reduction.

308. The method of claim 306, wherein said error reduction comprises:

a) exposing the construction oligonucleotides or the polynucleotide construct to a single stranded nuclease under conditions that permit cleavage of the construction oligonucleotides or polynucleotide construct having at least one mismatch by the nuclease; and

b) separating full length copies of the construction oligonucleotides or polynucleotide construct from cleaved copies of the construction oligonucleotides or polynucleotide construct.

309. The method of claim 308, wherein full length copies of the construction oligonucleotides or polynucleotide construct are separated from cleaved copies of the construction oligonucleotides or polynucleotide construct by size separation.

310. The method of claim 306, wherein the error reduction comprises:

a) exposing the construction oligonucleotides or the polynucleotide construct to a single stranded nuclease under conditions that permit cleavage of the construction oligonucleotides or the polynucleotide construct having at least one mismatch by the nuclease;

b) subjecting the digested construction oligonucleotides polynucleotide construct to a round of denaturation and renaturation; and

c) incubating the digested construction oligonucleotides or polynucleotide construct under chain extension, or chain extension and ligation, conditions thereby reforming the full length construction oligonucleotides or the full length polynucleotide construct.

311. A method for assembling at least one polynucleotide construct having a predefined sequence, comprising:

a) providing a pool of construction oligonucleotides comprising partially overlapping sequences that define the sequence of said polynucleotide construct;

wherein said pool comprises a plurality of populations of construction oligonucleotides, each population collectively having a defined consensus sequence but in which no more than 10 percent of said construction oligonucleotides in a given population have the identical sequence; and

b) providing conditions promoting;

i) hybridization of complementary, overlapping sequences of said construction oligonucleotides;

ii) ligation, chain extension, or chain extension and ligation, of hybridized construction oligonucleotides to form a polynucleotide construct;

iii) error reduction to provide a polynucleotide construct having said predetermined sequence.