De novo enzymatic production of nucleic acid molecules
The present invention relates to methods for making a nucleic acid molecule and methods for ligating oligonucleotides. The method includes ligating a first at least partially double-stranded oligonucleotide that has a first and second single-stranded overhang to a second at least partially double-stranded oligonucleotide that has a recognition site for a type IIS restriction enzyme, a modification allowing the oligonucleotide to be coupled to a surface, and a single-stranded overhang. The ligation product can be cleaved with a type IIS restriction enzyme, thereby releasing an elongated fragment having two single-stranded overhangs. These steps can be repeated by using the elongated fragment in a subsequent ligation to another at least partially double-stranded oligonucleotide that has a type IIS restriction enzyme recognition site.
1. A method for the manufacture of a nucleic acid molecule comprising the following steps:
(a) providing a first at least partially double-stranded oligonucleotide, whereby the oligonucleotide comprises a first single-stranded overhang, and a first modification allowing the oligonucleotide to be immobilised to a surface, wherein the first modification comprises a second single-stranded nucleotide overhang, wherein the first oligonucleotide is a first part of a nucleic acid to be manufactured,
(b) providing a second at least partially double-stranded oligonucleotide, which comprises a recognition site for a first type IIS restriction enzyme which cuts outside its recognition site, a second modification allowing the oligonucleotide to be coupled to a surface and a single-stranded overhang, wherein the second oligonucleotide is a second part of a nucleic acid to be manufactured,
(c) ligating the first oligonucleotide and the second oligonucleotide via the first single-stranded overhang of the first oligonucleotide and the single-stranded overhang of the second oligonucleotide, generating a first ligation product, whereby the first ligation product comprises the first modification allowing the first ligation product to be immobilised to a surface, wherein the first modification of the first ligation product is the second single-stranded nucleotide overhang of the first oligonucleotide,
(d) cutting the first ligation product with the first type IIS restriction enzyme thus releasing
an elongated first at least partially double-stranded oligonucleotide having a first and a second single-stranded overhang, whereby the first single-stranded overhang is generated through the cutting of the restriction enzyme and whereby the second single-stranded overhang is the second single-stranded nucleotide overhang of the first at least partially double-stranded oligonucleotide of step (a), and
a truncated second at least partially double-stranded oligonucleotide;
(e) immobilising the truncated second at least partially double stranded oligonucleotide of step d), the unreacted second at least partially double-stranded oligonucleotide and/or the uncut first ligation product via the second modification to a surface;
(f) repeating steps (a) to (e) at least once, whereby the elongated first at least partially double-stranded oligonucleotide of step (d) serves as the first at least partially double-stranded oligonucleotide in step (a), and is further elongated.
2. The method of claim 1 , comprising the following step ca) immobilising the first ligation product to a surface via the first modification comprising the single-stranded overhang.
3. The method of claim 2 , wherein the surface comprises a nucleic acid having a single-stranded stretch which is at least partially complementary to the first modification of the first ligation product comprising the single-stranded overhang.
4. The method of claim 1 , 2 or 3 , comprising the following step cb) washing the immobilised first elongation product; and cc) releasing the immobilised first elongation product from the surface.
5. The method of claim 4 , wherein the length of the first single-stranded overhang of the first at least partially complementary oligonucleotide has a length of 1, 2, 3, 4 or 5 nucleotides.
6. The method of claim 5 , wherein the first modification comprising the second single-stranded overhang of the first oligonucleotide allows for a stable hybridisation to the single-stranded stretch of the nucleic acid comprised on the surface.
7. The method of claim 6 , wherein the hybridisation is stable under the reaction conditions of step cb).
8. The method of claim 7 , wherein the modification comprising the second single-stranded overhang of the first oligonucleotide has a length from about 5 to 20 nucleotides, from about 10 to 20 nucleotides, from about 15 to 18 nucleotides, from about 5 to 10 nucleotides and from about 6 to 8 nucleotides, depending on the nature of the nucleotides.
9. The method of claim 1 , wherein the second modification of the second at least partially double-stranded oligonucleotide is a biotin modification.
10. The method of claim 9 , wherein the immobilisation of step e) occurs via interaction of the biotin and the surface, whereby the surface preferably comprises a biotin interaction group.
11. The method of claim 10 , wherein the biotin interaction group is selected from the group comprising avidin, streptavidin, extravidin, mutants of each thereof and synthetic biotin binding sites.
12. The method of claim 11 , wherein a part of the nucleic acid to be manufactured is part of the elongated first at least partially double-stranded oligonucleotide.
13. The method of claim 12 , wherein steps a) to e) are repeated at least once, whereby the nucleotides transferred from the second and any further at least partially double-stranded oligonucleotides provided in step b) to the first at least partially double-stranded oligonucleotides are the nucleic acid to be manufactured or a part thereof.