Method and system for the generation of large double stranded DNA fragments
First Claim
1. A method for the generation of a long double stranded DNA target sequence comprising:
- (a) synthesizing a set of oligonucleotides that contain sections of the target sequence, each oligonucleotide attached to a support by a cleavable linker;
(b) cleaving the linker to release selected oligonucleotides in a desired sequence, bringing the released oligonucleotides together, and joining selected oligonucleotides to form a set of subsequences which are parts of the desired target sequence; and
(c) assembling the subsequences to form the desired target sequence.
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Abstract
Synthesis of long chain molecules such as DNA is carried out rapidly and efficiently to produce relatively large quantities of the desired product. The synthesis of an entire gene or multiple genes formed of many hundreds or thousands of base pairs can be accomplished rapidly and, if desired, in a fully automated process requiring minimal operator intervention, and in a matter of hours, a day or a few days rather than many days or weeks. Production of a desired gene or set of genes having a specified base pair sequence is initiated by analyzing the specified target sequence and determining an optimal set of subsequences of base pairs that can be assembled to form the desired final target sequence. The set of oligonucleotides are then synthesized utilizing automated oligonucleotide synthesis techniques. The synthesized oligonucleotides are subsequently selectively released from the substrate and used in a sequential assembly process.
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Citations
25 Claims
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1. A method for the generation of a long double stranded DNA target sequence comprising:
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(a) synthesizing a set of oligonucleotides that contain sections of the target sequence, each oligonucleotide attached to a support by a cleavable linker;
(b) cleaving the linker to release selected oligonucleotides in a desired sequence, bringing the released oligonucleotides together, and joining selected oligonucleotides to form a set of subsequences which are parts of the desired target sequence; and
(c) assembling the subsequences to form the desired target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
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11. A method for the generation of nucleotides having a desired sequence comprising:
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(a) synthesizing a set of double stranded nucleotides that are intended to contain the desired sequence;
(b) carrying out coincidence filtering error correction on the nucleotides by passing double stranded nucleotides through a filter that binds DNA duplexes containing mismatched bases while allowing error free duplexes to pass through. - View Dependent Claims (12, 13, 14, 15, 16)
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17. A method for the generation of a long double stranded DNA target sequence comprising:
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(a) synthesizing a set of oligonucleotides that contain sections of the target sequence, each oligonucleotide attached to a support by a cleavable linker, wherein synthesizing is carried out in a maskless array synthesizer having a reaction chamber in which DNA synthesis reactions are performed on the support with an active surface in which arrays of different oligonucleotides are formed, a flow cell enclosing the active surface of the support and having ports for supplying reagents into the flow cell which can be flowed over the active surface of the support, a DNA synthesizer reagent supply connected to supply reagents to the flow cell, and an image former for providing a high precision, array light image projected onto the support active surface;
(b) cleaving the linker to release selected oligonucleotides in a desired sequence, bringing the released oligonucleotides together, and joining selected oligonucleotides to form a set of subsequences which are parts of the desired target sequence; and
(c) assembling the subsequences to form the desired target sequence. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25)
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Specification