Method for the manufacture of nucleic acid molecules

• US 7,695,906 B2
• Filed: 10/17/2003
• Issued: 04/13/2010
• Est. Priority Date: 10/18/2002
• Status: Active Grant

• Current Assignee: Morphosys Ag
• Original Assignee: Sloning BioTechnology GmbH
• Inventors: Waldmann, Thomas, O'Connell, Timothy, Schwer, Heinz, Schatz, Octavian
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Thomas; David C
• Application Number:
  US10/531,556
• Publication Number:
  US 20060194202A1
• Time in Patent Office: 2,370 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The present invention is related to a method for the manufacture of a nucleic acid molecule and compounds used therefore. The invention further provides a method of ligating, cleaving and immobilising oligonucleotides in order to manufacture nucleic acid molecules. The invention includes the steps wherein a first and second at least partially double-stranded oligonucleotides are ligated via their respective single-stranded overhangs. The ligation product may be immobilised to the surface via the modification that is provided on the first oligonucleotide. The immobilised ligation product is cleaved with the first type IIS restriction enzyme therein releasing an elongated oligonucleotide having an overhang. The elongated oligonucleotide may further be combined and ligated with a further at least partially double-stranded oligonucleotide to form a further ligated product that may be cleaved with a type IIS restriction enzyme releasing an elongated oligonucleotide having an overhang. The steps may be further repeated in various combinations.

Claims

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

(a) providing a first at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be coupled to a surface, whereby the oligonucleotide comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, and which oligonucleotide comprises a single-stranded overhang;

(b) providing a second at least partially double-stranded oligonucleotide whereby the oligonucleotide comprises a recognition site or a part thereof or a sequence which is complementary thereto, for a second type IIS restriction enzyme which cuts outside its recognition site, and which second oligonucleotide comprises a single-stranded overhang;

(c) ligating the first and the second oligonucleotide via their overhangs generating a first ligation product, wherein the first and second oligonucleotides are not attached to a surface during the ligation;

(d) immobilising the first ligation product of step (c) to the surface via the modification;

(e) cutting the immobilised ligation product with the first type IIS restriction enzyme thus releasing an elongated oligonucleotide having an overhang;

(f) combining the elongated oligonucleotide with a further at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be coupled, to a surface, whereby the further oligonucleotide comprises a recognition site for a further type IIS restriction enzyme which cuts outside its recognition site and which oligonucleotide comprises a single-stranded overhang, and ligating the elongated second oligonucleotide and the further at least partially double-stranded oligonucleotide via their overhangs forming a further ligation product;

(g) immobilising the further ligation product to a surface via the modification;

(h) cutting the further ligation product with the further type IIS restriction enzyme releasing an elongated oligonucleotide having an overhang; and

(i) optionally, repeating steps f) to h).

3. The method according to claims 1, or 2, wherein the overhang is a 5′

-overhang or a 3′

-overhang.

4. The method according to claim 3, wherein the overhang is selected from the group consisting a one-nucleotide overhang, a two nucleotides overhang, a three-nucleotides overhang, a four nucleotides overhang, a five-nucleotides overhang, a six-nucleotides overhang and a seven-nucleotides overhang.

5. The method according to claim 4, wherein the elongated oligonucleotide is transferred to a new reaction vessel where it is combined with the further oligonucleotide.

6. The method according to claim 5, wherein the at least partially double-stranded oligonucleotide comprises a constant region and a variable region whereby the constant region contains a recognition site for a type IIS restriction enzyme, and the variable region contains a nucleic acid sequence which corresponds to a part of the nucleic acid sequence of the nucleic acid molecule to be manufactured.

2. A method for the manufacture of a nucleic acid molecule comprising the steps of:

(a) providing a first at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be coupled to a surface, whereby the oligonucleotide comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, and which oligonucleotide comprises a single-stranded overhang;

(b) providing a second at least partially double-stranded oligonucleotide whereby the oligonucleotide comprises a recognition site or a part thereof or a sequence which is complementary thereto, for a second type IIS restriction enzyme which cuts outside its recognition site, and which second oligonucleotide comprises a single-stranded overhang;

(c) ligating the first and the second oligonucleotide via their overhangs generating a first ligation product, wherein the first and second oligonucleotides are not attached to a surface during the ligation;

(d) cutting the ligation product with the first type IIS restriction enzyme thus generating an elongated oligonucleotide having an overhang and a shortened first oligonucleotide;

(e) immobilising the shortened first oligonucleotide on a surface via the modification;

(f) providing a further at least partially double-stranded oligonucleotide which has a modification allowing the further oligonucleotide to be coupled to a surface, whereby the further oligonucleotide comprises a recognition site for a further type IIS restriction enzyme which cuts outside its recognition site and which oligonucleotide comprises a single-stranded overhang;

(g) combining the elongated oligonucleotide with the further oligonucleotide and ligating the elongated oligonucleotide and the further oligonucleotide via their overhangs forming a further ligation product;

(h) cutting the further ligation product with the further type IIS restriction enzyme generating an elongated oligonucleotide having an overhang and a shortened further oligonucleotide; and

(i) optionally, repeating steps e) to h).

7. A method for the manufacture of a nucleic acid molecule comprising the steps of:

(a) providing a first at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be coupled to a surface, whereby the oligonucleotide comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, and which oligonucleotide comprises a single-stranded overhang, and whereby the oligonucleotide comprises a part of the nucleic acid molecule to be manufactured;

(b) immobilizing the first oligonucleotide on a surface;

(c) cutting the first oligonucleotide with the first type IIS restriction enzyme releasing a double stranded oligonucleotide having a single stranded overhang at each end and being a part of the nucleic acid molecule to be manufactured; and

(d) combining the double stranded oligonucleotide of step c) with a second at least partially double-stranded oligonucleotide which has a modification allowing the oligonucleotide to be coupled to a surface, whereby the oligonucleotide contains a recognition site for a second type IIS restriction enzyme which cuts outside its recognition site, and which oligonucleotide further comprises a single-stranded overhang and a part of the nucleic acid molecule to be manufactured, and ligating the double-stranded oligonucleotide of step c) with the second oligonucleotide, wherein the double-stranded oligonucleotide of step c) and the second oligonucleotide are not attached to a surface during the ligation;

whereby the overhang of the second oligonucleotide is essentially complementary to the overhang of the double stranded oligonucleotide of step c).

8. The method according to claim 7, wherein the overhang generated upon cutting the first oligonucleotide with the first type IIS restriction enzyme is essentially complementary to the overhang of the second at least partially double stranded oligonucleotide.

9. A method for the manufacture of a nucleic acid molecule comprising the following steps:

(a) providing a first ligation product, whereby the first ligation product consists of a first oligonucleotide moiety comprising a recognition site for a first type IIS restriction enzyme, a second oligonucleotide moiety comprising a recognition site for a second type IIS restriction enzyme and a third oligonucleotide moiety, whereby the third oligonucleotide moiety is a part of the nucleic acid molecule to be manufactured, and whereby the first and the second type IIS restriction enzymes each generate an overhang, whereby the overhang generated by the first type IIS restriction enzyme has a length which is different from the length of the overhang generated by the second type IIS restriction enzyme;

(b) providing a second ligation product, whereby the second ligation product consists of a first oligonucleotide moiety comprising a recognition site for a third type IIS restriction enzyme, a second oligonucleotide moiety comprising a recognition site for a fourth type IIS restriction enzyme and a third oligonucleotide moiety, whereby the third oligonucleotide moiety is a part of the nucleic acid molecule to be manufactured, and whereby the third and the fourth type IIS restriction enzyme each generate an overhang, whereby the overhang generated by the third type IIS restriction enzyme has a length which is different from the length of the overhang generated by the fourth type IIS restriction enzyme;

(c) cutting the first ligation product with the second restriction enzyme generating a first cut ligation product and cutting the second ligation product with the fourth restriction enzyme generating a second cut ligation product;

(d) providing a third at least partially double-stranded oligonucleotide and ligating the third oligonucleotide with the first cut ligation product, wherein the first cut ligation product and the third oligonucleotide are not attached to a surface during the ligation, whereby the third oligonucleotide comprises an overhang which is complementary to the overhang of the first cut ligation product generated in step c) and whereby the third oligonucleotide comprises a recognition site for a fifth IIS restriction enzyme;

(e) providing a fourth at least partially double-stranded oligonucleotide and ligating the fourth oligonucleotide to the second cut ligation product, wherein the second cut ligation product and the fourth oligonucleotide are not attached to a surface during the ligation, whereby the fourth oligonucleotide comprises an overhang which is complementary to the overhang of the second ligation product generated in step c) and whereby the fourth oligonucleotide comprises a recognition site for a sixth type IIS restriction enzyme;

(f) immobilising the ligation product of step d) and step e) on a surface by means of a modification of the third oligonucleotide and the fourth oligonucleotide;

(g) cutting the immobilised ligation product of step d) with the fifth type IIS restriction enzyme releasing an oligonucleotide;

(h) cutting the immobilised ligation product of step e) with the third type IIS restriction enzyme; and

(i) combining and ligating the oligonucleotide released according to step g) with the immobilised reaction product of step h), whereby the overhang generated by the first and the third restriction enzyme is complementary to the overhang generated by the fifth and sixth restriction enzyme.

10. The method according to claim 9, wherein the first and the third restriction enzyme are identical and/or the second and the fourth restriction enzyme are identical and/or the fifth and the sixth restriction enzyme are identical.

11. The method according to claim 10, wherein the first and the third restriction enzyme and the fifth and the sixth restriction enzyme are each a restriction enzyme generating a four-nucleotide overhang at the 3′

or 5′

end.

12. The method according to claim 11, wherein the second and the third restriction enzyme is a restriction enzyme creating an overhang having a length which is selected from the group consisting of one, two, three, four, five and six nucleotides.

13. The method according to claim 12, wherein the first and the second restriction enzyme is Esp3I or Eco31I and the fifth and the sixth restriction enzyme is Eco3 11 or Esp3I.

14. The method according to claim 13, wherein the ligation product of step i) is used as a first ligation product and/or a second ligation product and steps a) to i) are repeated one or several times.

15. The method according to claim 14, wherein the third moiety is arranged between the moieties of the oligonucleotides containing the restriction site for the type IIS restriction enzymes.

16. The method according to claim 15, wherein the first and he second ligation products are provided in separate reaction vessels.