METHOD OF ON-CHIP NUCLEIC ACID MOLECULE SYNTHESIS

• US 20140309142A1
• Filed: 04/16/2013
• Published: 10/16/2014
• Est. Priority Date: 04/16/2012
• Status: Abandoned Application

• Current Assignee: Jingdong Tian
• Original Assignee: Jingdong Tian
• Inventors: TIAN, Jingdong
• Application Number:
  US13/864,100
• Publication Number:
  US 20140309142A1
• Time in Patent Office: Days
• US Class Current: 506/12
• 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 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• 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

Abstract

A method of synthesizing a nucleic acid molecule, such as a gene, on a substrate or microchip is described. In particular, a method for synthesizing, amplifying, and assembling DNA oligonucleotides into a nucleic acid molecule or gene product, on a single substrate or microchip is described. Also described are a method of correcting a sequence error in a synthesized nucleic acid molecule, as well as a method for synthesizing and screening a library of codon variants to identify a nucleic acid molecule with an optimized level of protein expression.

Claims

1. A method of synthesizing a nucleic acid molecule having a target sequence, comprising:

(1) obtaining a substrate having a chamber comprising a plurality of immobilized oligonucleotides for the synthesis of the target sequence,

(2) adding to the chamber a reaction mixture comprising dNTPs, a primer, a strand-displacing polymerase, a nicking endonuclease, a heat-stable DNA polymerase, and a buffer;

(3) amplifying the plurality of oligonucleotides to obtain free amplified oligonucleotides by a nicking strand displacement amplification reaction in the chamber containing the reaction mixture; and

(4) assembling the free amplified oligonucleotides by a polymerase cycling assembly reaction to obtain the nucleic acid molecule;

wherein step (4) is conducted in the chamber without the need for a buffer change after step (3).

2. The method according to claim 1, further comprising amplifying the nucleic acid molecule obtained in step (3) by a polymerase chain reaction (PCR) amplification reaction to obtain an amplified nucleic acid molecule, and purifying the amplified nucleic acid molecule.

3. The method according to claim 1, wherein

each of the plurality of oligonucleotides comprises a universal adaptor sequence at the 3′

end of the oligonucleotide and a portion of the target sequence or a portion of a sequence complementary to the target sequence;

the universal adaptor sequence anchors the oligonucleotide to the substrate surface; and

the primer comprises a universal primer complementary to the universal adaptor sequence, the universal primer comprises a nucleotide sequence that is recognized and cut by the nicking endonuclease.

4. The method according to claim 3, wherein the portion of the target sequence or its complementary sequence comprises about 48 to 150 bases in length;

and the universal adaptor sequence comprises about 15-35 bases in length.

5. The method according to claim 3, wherein the universal adaptor comprises a Nt.BstNBI recognition site;

and the reaction mixture comprises the universal primer, Nt.BstNBI, Bst DNA polymerase, large fragment, Phusion polymerase and a buffer.

6. The method according to claim 1, wherein a plurality of nucleic acid molecules are synthesized in each of a plurality of chambers, and the plurality of chambers are on a microchip.

7. The method of claim 1, further comprising transforming a cell with the nucleic acid molecule obtained from step (3).

8. The method according to claim 2, further comprising correcting a sequence error in the amplified and purified nucleic acid molecule, comprising:

(1) heating and subsequently cooling a plurality of nucleic acid molecules synthesized according to a method of the present invention, thereby forming one or more heteroduplexes, wherein the heteroduplex comprises one or more mismatch sites resulting from the errors;

(2) contacting the one or more heteroduplexes with a mismatch-specific endonuclease under conditions for effective cleavage of the one or more heteroduplexes at the one or more mismatch sites, thereby obtaining cleaved fragments; and

(3) contacting the cleaved fragments with a DNA polymerase having 3′

-5′

exonuclease activity under conditions for an overlap extension polymerase chain reaction amplification, thereby producing a plurality of nucleic acid molecules free of the one or more errors.

9. The method of claim 8, wherein the mismatch-specific endonuclease is a CEL endonuclease.

10. The method of claim 8, wherein the CEL endonuclease is a CEL II endonuclease from celery, and the DNA polymerase is Phusion polymerase.

11. The method of claim 8, further comprising transforming a cell with the nucleic acid molecule obtained from step (3).

12. The method of claim 8, further comprising repeating steps (1) to (3) of claim 8.

13. A method for screening a library of codon variants to obtain a nucleic acid sequence for optimized protein expression, the method comprising:

(1) synthesizing the library of codon variants using the method of claim 1;

(2) amplifying the library by a polymerase chain reaction (PCR) amplification reaction;

(3) operably linking the library of codon variants to a reporter gene sequence to obtain a library of reporter constructs;

(4) introducing the library of reporter constructs into a host cell; and

(5) measuring the expression of the reporter gene sequence from the host cell, thereby identifying the nucleic acid sequence for optimized protein expression.

14. The method according to claim 13, further comprising sequencing the identified nucleic acid sequence.

15. A method of on-chip synthesis of a gene comprises:

(1) obtaining a microchip comprising multiple chambers, each chamber comprising a plurality of immobilized oligonucleotides for the synthesis of a target sequence, wherein the target sequence comprises a fragment of the gene;

(2) adding to each of the chambers a reaction mixture comprising dNTPs, a primer, a strand-displacing polymerase, a nicking endonuclease, a heat-stable DNA polymerase, and a buffer;

(3) amplifying the plurality of oligonucleotides to obtain free amplified oligonucleotides by a nicking strand displacement amplification reaction in each of the chambers containing the reaction mixture;

(4) assembling the free amplified oligonucleotides to obtain the target sequence by a polymerase cycling assembly reaction, wherein step (4) is conducted in each of the chambers without the need for a buffer change after step (3).

(5) amplifying the target sequence from step (4) by a polymerase chain reaction (PCR) in each of the chambers;

(6) assembling the amplified target sequences from all chambers into a synthesized gene sequence; and

(7) correcting a sequence error in the synthesized gene sequence, comprising;

i. forming a heteroduplex comprising the synthesized gene sequence, the heteroduplex comprising one or more mismatch sites resulting from the sequence error;

ii. contacting the heteroduplex with a mismatch-specific endonuclease under conditions such that the heteroduplex is cleaved at the mismatch sites to obtain cleaved fragments of the gene; and

iii. contacting the cleaved fragments with a DNA polymerase having 3′

-5′

exonuclease activity under conditions for an overlap extension polymerase chain reaction amplification, thereby producing the gene sequence free of the sequence error.

16. The method according to claim 15, wherein

each of the plurality of oligonucleotides comprises a universal adaptor sequence at the 3′

end of the oligonucleotide and a portion of the target sequence or a portion of a sequence complementary to the target sequence;

the universal adaptor sequence anchors the oligonucleotide to the substrate surface; and

the primer comprises a universal primer complementary to the universal adaptor sequence, the universal primer comprises a nucleotide sequence that is recognized and cut by the nicking endonuclease.

17. A kit for performing on chip gene synthesis, the kit comprising:

(1) a universal primer comprising a nucleotide sequence that is recognized and cut by a nicking endonuclease,

(2) the nicking endonuclease;

(3) a strand displacement DNA polymerase;

(4) a DNA polymerase; and

(5) instructions on using the kit for synthesizing a nucleic acid molecule.

18. The kit of claim 17, wherein the DNA polymerase has 3′

-5′

exonuclease activity, and the kit further comprises a mismatch-specific endonuclease and additional instructions on enzymatic correction of sequence errors in the synthesized nucleic acid molecule.

19. The kit of claim 18, wherein the mismatch-specific endonuclease is a CEL endonuclease and the DNA polymerase is Phusion polymerase.