Programmable oligonucleotide synthesis
The invention relates to methods for the preparation of synthetic nucleic acids, such as double-stranded nucleic acids. The methods involve the preparation of a multiplicity of different nucleic acid fragments by solid-phase synthesis, and the joining together of at least two of the multiplicity of the nucleic acid fragments by binding to one another or by covalent linkage. In the methods at least some of the nucleic acid fragments have a high AT content and are used in an increased amount relative to other fragments for the joining step.
1. A method of producing double stranded synthetic nucleic acids comprising:
(a) producing a multiplicity of different nucleic acid fragments by solid-phase synthesis, at various positions of a solid reaction support, and
(b) joining at least two nucleic acid fragments from step (a) by binding them to one another by a specific hybridization reaction and/or by covalent linkage,
wherein at least some nucleic acid fragments have a higher AT content relative to other fragments in step (b) and each of said higher AT containing fragments is used in a higher concentration relative to other fragments in step (b); thereby producing the double stranded synthetic nucleic acids.
2. The method of claim 1 further comprising producing an RNA molecule from the double stranded synthetic nucleic acids using an RNA polymerase.
3. The method of claim 2 further comprising using the RNA molecule produced for the modulation of gene expression by RNAi and/or antisense methods.
4. The method of claim 1 wherein the synthetic nucleic acid encodes a polypeptide and wherein the method further comprises producing the polypeptide from the nucleic acid.
5. The method of claim 1 wherein the high AT content is more than 60%.
6. The method of claim 1 wherein the high AT content is more than 65%.
7. The method of claim 1 wherein the at least two nucleic acid fragments from step (a) are joined by a specific hybridization reaction.
8. The method of claim 1 wherein the at least two nucleic acid fragments of step (a) have a length of 20-300 nucleotides.
9. The method of claim 8 wherein the at least two nucleic acid fragments of step (a) have a length of 25-100 nucleotides.
10. The method of claim 8 wherein the solid reaction support is selected from the group consisting of: one or more beads, one or more particles, one or more chips, and a microfluidic reaction support.
11. The method of claim 10 wherein the solid reaction support is one or more beads or one or more chips.
12. The method of claim 1 wherein the solid reaction support is a microfluidic reaction support.
13. The method of claim 1 wherein the joining is by covalent linkage and the synthetic nucleic acid is a gene, gene cluster, chromosome, genome, or a fragment thereof.
14. The method of claim 1 wherein the solid reaction support is selected from the group consisting of: one or more beads, one or more particles, one or more chips, and a microfluidic reaction support.
15. The method of claim 14 wherein the solid reaction support is one or more beads or one or more chips.