Method for the synthesis of DNA fragments

• US 8,137,906 B2
• Filed: 01/20/2006
• Issued: 03/20/2012
• Est. Priority Date: 06/07/1999
• Status: Expired due to Term

• Current Assignee: Morphosys Ag
• Original Assignee: Sloning BioTechnology GmbH
• Inventors: Schatz, Octavian
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US11/336,198
• Publication Number:
  US 20060115850A1
• Time in Patent Office: 2,251 Days
• Field of Search: 435/6, 435/91.1, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The invention relates to a method that can be carried out in parallel and automated for the production of any nucleic acid, comprising the following steps: a) coupling an oligonucleotide to a solid matrix b) adding an additional oligonucleotide c) ligating the oligonucleotides from steps a) and b) in one orientation d) removing excess reactants and enzymes from the reaction preparation e) cleaving the ligation product from step c) with a restriction enzyme that cleaves outside the recognition sequence, whereby cleavage occurs in the oligonucleotide from step a) or in the oligonucleotide from step b) f) separating the reaction mixture from the lengthened or shortened oligonucleotide from step a) that is obtained in step e) g) repeating steps b) to f) at least once h) successive sequence-independent linkage of the fragments obtained after performing steps a) to g) until the desired product is obtained.

Claims

1. A method for producing a synthesized nucleic acid molecule comprising the steps of:

(a) providing a double-stranded first oligonucleotide which is prepared by the following steps;

aa) coupling one end of a first modified oligonucleotide comprising a double-stranded nucleic acid to a surface of a first solid matrix wherein the first modified oligonucleotide contains a recognition sequence for a first type IIS restriction enzyme which cleaves the downstream sequence of the recognition sequence in the first modified oligonucleotide,

ab) providing a double-stranded second oligonucleotide that is different from the first modified oligonucleotide of step aa), wherein the second oligonucleotide is partially self-complementary, and contains a loop and a recognition sequence for a second type IIS restriction enzyme which cleaves the downstream sequence of the recognition sequence in the second oligonucleotide, wherein the recognition sequence of the first type II restriction enzyme in the first modified oligonucleotide and the recognition sequence of the second type IIS restriction enzyme in the second oligonucleotide are different,

ac) ligating the first modified oligonucleotide on the surface of the first solid matrix and the second oligonucleotide from steps aa) and ab) and forming a first ligation product,

ad) removing the first modified oligonucleotide and the second oligonucleotide from steps aa) and ac) that are not coupled or ligated,

ae) cleaving the first ligation product obtained from step ad) with the second type IIS restriction enzyme whereby the cleavage occurs in the nucleic acid sequence of the second oligonucleotide of the first ligation product and resulting in a first elongated oligonucleotide comprising the recognition sequence of the first type IIS restriction enzyme and a first shorter oligonucleotide, wherein the first ligation product from step ac) is double-stranded,

af) separating the second type IIS restriction enzyme and the first shorter oligonucleotide from the first elongated oligonucleotide obtained in step ae),

ag) providing a double-stranded third oligonucleotide that is different from the first elongated oligonucleotide, wherein the third oligonucleotide is partially self-complementary, and contains a loop and a recognition sequence for a third type IIS restriction enzyme which cleaves the downstream sequence of the recognition sequence in the third oligonucleotide, wherein the recognition sequence for the first type IIS restriction enzyme in the elongated oligonucleotide and the recognition sequence for the third type IIS restriction enzyme in the third oligonucleotide are different,

ah) ligating the first elongated oligonucleotide obtained from steps af) and the third oligonucleotide of step ag) and forming a second ligation product,

ai) removing the first elongated oligonucleotide and the third oligonucleotide from step ah) that are not ligated,

aj) cleaving the second ligation product obtained from step ai) with the third type IIS restriction enzyme whereby the cleavage occurs in the nucleic acid sequence of the third oligonucleotide of the second ligation product and resulting in a second elongated oligonucleotide comprising the recognition sequence of the first type IIS restriction enzyme and a second shorter oligonucleotide, wherein the second ligation product from step ah) is double-stranded,

ak) separating the third type IIS restriction enzyme and the second shorter oligonucleotide from the second elongated oligonucleotide obtained in step aj), wherein the second elongated oligonucleotide is the first oligonucleotide;

(b) providing a double-stranded fourth oligonucleotide which is prepared by the following steps;

ba) coupling one end of a second modified oligonucleotide comprising a double-stranded nucleic acid to a surface of a second solid matrix wherein the second modified oligonucleotide contains a recognition sequence for a fourth type IIS restriction enzyme which cleaves the downstream sequence of the recognition sequence in the second modified oligonucleotide,

bb) providing a double-stranded fifth oligonucleotide that is different from the second modified oligonucleotide of step ba), wherein the fifth oligonucleotide is partially self-complementary, and contains a loop and a recognition sequence for a fifth type IIS restriction enzyme which cleaves the downstream sequence of the recognition sequence in the fifth oligonucleotide, wherein the recognition sequence for the fourth type IIS restriction enzyme in the second modified oligonucleotide and the recognition sequence for the fifth type IIS restriction enzyme in the fifth oligonucleotide are different,

bc) ligating the second modified oligonucleotide on the surface of the second solid matrix and the fifth oligonucleotide from steps ba) and bb) and forming a third ligation product,

bd) removing the second modified oligonucleotide and the fifth oligonucleotide from steps ba) and bc) that are not coupled or ligated,

be) cleaving the third ligation product obtained from step bd) with the fifth type IIS restriction enzyme whereby the cleavage occurs in the fifth oligonucleotide of the third ligation product and resulting in a third elongated oligonucleotide comprising the recognition sequence of the fourth type IIS restriction enzyme and a third shorter oligonucleotide, wherein the third ligation product from step bc) is double-stranded,

bf) separating the fifth type IIS restriction enzyme and the third shorter oligonucleotide from the third elongated oligonucleotide obtained in step be),

bg) providing a double-stranded sixth oligonucleotide that is different from the third elongated oligonucleotide, wherein the sixth oligonucleotide is partially self-complementary, and contains a loop and a recognition sequence for a sixth type IIS restriction enzyme which cleaves the downstream sequence of the recognition sequence in the sixth oligonucleotide, wherein the recognition sequence for the fourth type IIS restriction enzyme in the third elongated oligonucleotide and the recognition sequence for the sixth type IIS restriction enzyme in the sixth oligonucleotide are different,

bh) ligating the third elongated oligonucleotide obtained from step bf) and the sixth oligonucleotide of step bg) and forming a fourth ligation product,

bi) removing the third elongated oligonucleotide and the sixth oligonucleotide from step bh) that are not ligated,

bj) cleaving the fourth ligation product obtained from step bi) with the sixth type IIS restriction enzyme whereby the cleavage occurs in the sixth oligonucleotide of the fourth ligation product and resulting in a fourth elongated oligonucleotide comprising the recognition sequence of the fourth type IIS restriction enzyme and a fourth shorter oligonucleotide, wherein the fourth ligation product from step bh) is double-stranded,

bk) separating the sixth type IIS restriction enzyme and the fourth shorter oligonucleotide from the fourth elongated oligonucleotide obtained in step bj), wherein the fourth elongated oligonucleotide is the fourth oligonucleotide;

(c) ligating the first oligonucleotide and the fourth oligonucleotide from steps (a) and (b) and forming a fifth ligation product,

(d) removing the first oligonucleotide and the fourth oligonucleotide from step (c) that are not ligated;

(e) cleaving the fifth ligation product obtained from step (d) with the first type IIS restriction enzyme or the fourth type IIS restriction enzyme, and resulting in a fifth elongated oligonucleotide and a fifth shorter oligonucleotide, wherein the fifth ligation product from step (c) is double-stranded; and

(f) separating and removing the first type IIS restriction enzyme or the fourth type IIS restriction enzyme and the fifth shorter oligonucleotide from the fifth elongated oligonucleotide obtained from step (e), wherein the fifth elongated oligonucleotide is retained, wherein the synthesized nucleic acid molecule is the fifth elongated oligonucleotide.

2. The method of claim 1, wherein further comprises performing an exonuclease and/or phosphatase reaction/reactions after step ac), bc), or (c).

3. The method of claim 2, wherein unreacted exonuclease and/or phosphatase are removed after the exonuclease and/or phosphatase reaction/reactions.

4. The method of claim 1, wherein the end of the first modified oligonucleotide from step aa) or the second modified oligonucleotide ba) that is not coupled to the surface of the first solid matrix or the second solid matrix contains a first part of a recognition sequence for a seventh type IIS restriction enzyme, and wherein the second oligonucleotide from step ab) or the fifth oligonucleotide from step bb) contains the second part of the recognition sequence for the seventh type IIS restriction enzyme.

5. The method of claim 1, wherein each of the first modified oligonucleotide and the second modified oligonucleotide comprises biotin, digoxigenin, fluorescein isothiocyanate, an amino compound or a succinyl ester.

6. The method as claimed in claim 1, wherein the first modified oligonucleotide from step aa) or the first oligonucleotide from step (a) and/or the second modified oligonucleotide from step ab) or the fourth oligonucleotide from step (b) have/has a loop.

7. The method of claim 6, wherein the loop of the first modified oligonucleotide from step aa) or the second modified oligonucleotide from step ba) comprises a modification.

8. The method of claim 1, wherein each of the first solid matrix and the second solid matrix is selected from the group consisting of a bead, a microscope slide, a DNA chip, a well of a microtitre plate and a test tube.

9. The method of claim 8, wherein each of the first solid matrix and the second solid matrix is a bead and said bead is made of a material selected from the group consisting of glass and polystyrene.

10. The method of claim 1, wherein each of the first solid matrix and the second solid matrix comprises streptavidin, an anti-digoxigenin antibody, or an anti-fluorescein isothiocyanate antibody.

11. The method of claim 1, wherein the end of the first modified oligonucleotide from step aa) or the end of the second modified oligonucleotide from step ba) that is not coupled to the surface of the first solid matrix or the second solid matrix has a single-stranded overhang which is mutually complementary to an end of the second oligonucleotide from step ab) or an end of the fifth oligonucleotide from step bb), and wherein the end of the first modified oligonucleotide from step aa) or the end of the second modified oligonucleotide from step ba) that is not coupled to the surface of the first solid matrix or the second solid matrix and the end of the second oligonucleotide from step ab) or the end of the fifth oligonucleotide from step bb) are ligated to each other in step ac) or bc).

12. The method of claim 11, wherein the single strand overhang is 1, 2, 3, 4, or 5 nucleotides long.

13. The method of claim 1, wherein the second oligonucleotide in step ab) or the fifth oligonucleotide in step bb) is a PCR product, a plasmid vector, a phage or viral DNA, an artificial chromosome or another synthetic DNA.