Synthesis of sequence-verified nucleic acids
The invention relates to methods and devices for preparing synthetic nucleic acids.
1. A method of retrieving sequence verified nucleic acids from a solid support comprising: providing a mixture of nucleic acid molecules, and
(1) generating a nucleic acid library by individualizing the mixture of nucleic acids on the solid support by physical separation of the individual nucleic acid molecules into defined locations;
(2) sequencing the individualized members of the nucleic acid library at the single molecule level without first performing a step of amplification, and assigning a sequence to each member of the library to produce a library of sequence-verified nucleic acid molecules at the defined locations;
(3) retrieving sequence-verified nucleic acid molecules from the solid support; and
(4) using the retrieved molecules in continuative steps/procedures to form a larger DNA molecule.
2. The method of claim 1 , wherein the method further comprises:
(a) isolating the sequence verified nucleic acid molecules of (3).
3. The method of claim 1 , wherein individualizing the mixture of nucleic acids comprises separation so that individual nucleic acids are present at a defined location on the solid support.
4. The method of claim 1 , wherein (4) comprises an amplification reaction of the individualized nucleic acids, wherein the amplification comprises emulsion PCR, bridge amplification, MegaPlex PCR, creation of polonies, or/and rolling circle amplification.
5. The method of claim 1 , wherein Step (2) comprises sequencing-by-synthesis or sequencing-by-ligation techniques.
6. The method of claim 1 , wherein retrieval Step (3) comprises physical retrieval, physical recovery and/or extraction.
7. The method of claim 4 , comprising isolating the solid support.
8. The method of claim 1 , wherein the retrieval in (3) further comprises enzymatic amplification of the nucleic acids presented on the solid support.
9. The method of claim 8 , comprising copying-off the nucleic acid molecules presented on the solid support by a polymerase.
10. The method of claim 1 , wherein the use of the retrieved molecules in continuative steps/procedures comprises the production of a bead-array or a micro-array.
11. The method of claim 10 , comprising the fabrication of a synthetic gene.
12. The method of claim 1 , further comprising performing (2) to (4) in a second cycle with a sub-population selected from the nucleic acid molecules obtained in (3) of a first cycle.
13. The method of claim 1 , where the defined locations on the solid support are identified by reference points in a microscopic picture of the solid support.
14. The method of claim 1 wherein the solid support is selected from the group consisting of: a particle, a bead, a microfluidic reaction support, a biochip, a DNA chip, a microtiter plate, a nanotiter plate, and a picotiter plate.
15. The method of claim 1 wherein the solid support is selected from the group consisting of: a microfluidic reaction support, a biochip, and a DNA chip.
16. The method of claim 15 wherein the solid support is a biochip.
17. The method of claim 1 wherein the method is an automated method.
18. The method of claim 1 , wherein the solid support is selected from the group consisting of glass slides, gels, polymers, capillaries, microfluidic carriers, membranes, porous carriers, plastics, silicon, ordered or chaotic pores, sponge structures, cubes, a 3 D matrix, emulsions, dendrimers, beads, particles, resins, metals, nano-particles and nanostructures or combinations thereof.
19. The method of claim 1 wherein retrieving sequence-verified nucleic acid molecules from the solid support comprises amplification of an individualized member of the nucleic acid library using a primer.
20. The method of claim 19 wherein the continuing steps comprise performing the polymerase chain reaction to amplify the individualized member of the nucleic acid library.