COMPOSITIONS AND METHODS FOR NUCLEIC ACID ASSEMBLY

• US 20160017394A1
• Filed: 07/15/2015
• Published: 01/21/2016
• Est. Priority Date: 07/15/2014
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Katzen, Federico, LIANG, Xiquan, Baek, Chang-Ho, Peng, Lansha
• Application Number:
  US14/800,384
• Publication Number:
  US 20160017394A1
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The present disclosure generally relates to compositions and methods for the assembly of nucleic acid molecules into larger nucleic acid molecules. Also provided are compositions and methods for seamlessly connection of nucleic acid molecules with high sequence fidelity.

Claims

1. A method for covalently linking two nucleic acid segments, the method comprising:

(a) incubating the two nucleic acid segments with an exonuclease under conditions that allow for digestion of termini of the two nucleic acid segments to form complementary single-stranded regions on each nucleic acid segment and hybridization of the complementary single-stranded regions, wherein each of the two nucleic acid segments comprises an exonuclease resistant region within 30 nucleotides of the end of the complementary terminus, and

(b) covalently connecting at least one strand of the hybridized termini formed in (a) resulting in the linkage of the two nucleic acid segments.

2. The method of claim 1, wherein steps (a) and (b) occur in the same tube.

3. The method of claim 1, wherein steps (a) and (b) occur in the same reaction mixture.

4. The method of claim 1, wherein the two or more nucleic acid segments are simultaneously contacted with an exonuclease and a ligase in step (a).

5. The method of claim 1, wherein the covalently linking of at least one strand of the hybridized termini formed in (a) is mediated by a ligase.

6. The method of claim 1, wherein three or more nucleic acid segments are covalently linked to each other.

7. The method of claim 1, wherein the two or more nucleic acid segments are covalently linked to one or more additional nucleic acid segments that do not contain exonuclease resistant regions.

8. The method of claim 1, wherein a replicable nucleic acid molecule is formed.

9. The method of claim 8, wherein the two or more nucleic acid segments are covalently linked form a circular nucleic acid molecule.

10. The method of claim 9, where the circular nucleic acid molecule contains one or more selection marker or origin of replication that is reconstituted by the linking of different nucleic acid segments.

11. The method of claim 9, wherein the circular nucleic acid molecule if formed from at least three nucleic acid segments.

12. The method of claim 8, wherein one or more enzyme contacting the nucleic acid molecule is partially or fully inactivated.

13. The method of claim 10, wherein inactivation of the one or more enzyme is inactivated by heating.

14. The method of claim 12, wherein the nucleic acid molecule is stored at −

20°

C. for at least two weeks after inactivation of the one or more enzyme.

15. A method for assembling a nucleic acid molecule, the method comprising:

(a) incubating a first nucleic acid segment with an exonuclease under conditions that allow for partial digestion of at least one terminus of the first nucleic acid segment to form a single-stranded region, wherein the first nucleic acid segment contains an exonuclease resistant region within 30 nucleotides of the at least one terminus,

(b) preparing a reaction mixture containing the digested first nucleic acid segment formed in (a) with an undigested second nucleic acid segment under conditions that allow for the hybridization of termini with sequence complementarity, and

(c) covalently connecting at least one strand of the hybridized termini formed in (b).

16. The method of claim 15, wherein the second nucleic acid segment of (b) contains no exonuclease resistant regions.

17. The method of claim 15, wherein at least one terminus of the second nucleic acid segment of (b) contains a single-stranded region with sequence complementarity to the single-stranded region of the first nucleic acid molecules formed in step (a).

18. The method of claim 15, wherein the exonuclease is a 5′

to 3′

exonuclease.

19. The method of claim 15, wherein two or more exonucleases are present in step (a).

20. The method of claim 15, wherein a functional exonuclease is present in step (b).

21. A method for assembling a nucleic acid molecule, the method comprising:

(a) incubating two or more nucleic acid segments with an exonuclease under conditions that allow for partial digestion of at least one terminus of each of the two or more nucleic acid segments to generate single-stranded termini, wherein at least two of the two or more nucleic acid segments contain an exonuclease resistant region within 30 nucleotides of at least one of their termini,

(b) preparing a reaction mixture containing the digested nucleic acid segments prepared in (a) with one or more undigested nucleic acid segment under conditions that allow for the hybridization of termini with sequence complementarity, wherein at least one of the one or more undigested nucleic acid segment has region of sequence complementarity with at least one single-stranded terminus formed in (a), and

(c) covalently connecting at least one strand of the hybridized termini formed in (b).