PCR-DIRECTED GENE SYNTHESIS FROM LARGE NUMBER OF OVERLAPPING OLIGODEOXYRIBONUCLEOTIDES
First Claim
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1. A PCR-directed method of synthesizing a gene of interest, wherein the method comprises the following steps:
- (a) determining an optimal concentration of a plurality of overlapping oligonucleotides;
(b) assembling the plurality of overlapping oligonucleotides at the determined optimal concentration by at least one cycle of assembly PCR to generate template DNA; and
(c) amplifying the template DNA with two separate and distinct outermost overlapping oligonucleotides by at least one cycle of amplification PCR.
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Abstract
The present invention provides methods of PCR-directed gene synthesis that may be used for all genes, including those with a high G+C content and/or a long sequence. The invention relates to methods of gene synthesis using overlapping oligonucleotides and polymerase chain reaction (PCR), wherein several PCR parameters, e.g., the concentration of overlapping oligonucleotides, the type of DNA polymerase used, and the number of PCR amplification cycles, are optimized. Additionally, the invention relates to oligonucleotide design that allows for increased protein expression of synthesized genes.
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19 Claims
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1. A PCR-directed method of synthesizing a gene of interest, wherein the method comprises the following steps:
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(a) determining an optimal concentration of a plurality of overlapping oligonucleotides; (b) assembling the plurality of overlapping oligonucleotides at the determined optimal concentration by at least one cycle of assembly PCR to generate template DNA; and (c) amplifying the template DNA with two separate and distinct outermost overlapping oligonucleotides by at least one cycle of amplification PCR. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification