Long nucleic acid sequences containing variable regions
AssigneeIntegrated Dna Technologies, Inc.
InventorsShawn Allen, Kristin Beltz, Scott Rose
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Abstract
This invention pertains to improved methods for the synthesis of long, double stranded nucleic acid sequences containing difficult to clone or variable regions.
Claims21
A method of constructing a double stranded DNA fragment or library, said method comprising incorporating sequences between clonal or non-clonal double stranded DNA fragments (gene blocks), the method comprising:
- a) forming a mixture comprised of a first gene block, a second gene block, and a bridging oligonucleotide set, said bridging oligonucleotide set comprising one or more bridging oligonucleotides, wherein each bridging oligonucleotide contains a first region that is hybridizable to a portion of the first gene block and a second region that is hybridizable to a portion of the second gene block;
- b) subjecting the mixture to reagents and conditions for PCR to assemble the gene blocks and bridge(s) thereby generating and optionally amplifying a double stranded DNA fragment or library, wherein the sequence generated is comprised of the first gene block, a bridge sequence of the bridging oligonucleotide(s), if any, that did not hybridize to a gene block, and the second gene block.
A method of constructing a double stranded DNA fragment or library, said method comprising incorporating sequences between clonal or non-clonal double stranded DNA fragments (gene blocks), the method comprising:
- a) forming a mixture comprised of more than two gene blocks, and a bridging oligonucleotide set, said bridging oligonucleotide set comprising one or more bridging oligonucleotides, and wherein each bridging oligonucleotide contains a first region that is hybridizable to a portion of one gene block and a second region that is hybridizable to a portion of another gene block wherein, when mixed together, a resulting product comprises successive gene blocks linked by bridging oligonucleotides;
- b) subjecting the mixture to reagents and conditions for PCR to assemble the gene blocks and bridge(s) and thereby generating and amplifying a double stranded DNA fragment or library, wherein the sequence generated is comprised of the first gene block, the bridge sequence of the bridging oligonucleotide(s), and the second gene block.
A kit for the manufacture of a double-stranded DNA fragment library, said kit comprising:
- (a) two or more gene blocks;
- and
- (b) one or more bridging oligonucleotide, wherein each bridging oligonucleotide contains a first region of 10-50 bases substantially complementary to a strand of a first gene block and a second region of 10-50 bases substantially complementary to a strand of a second gene block, and wherein the bridging oligonucleotide contains 1-30 degenerate bases.
The kit of claim 20 wherein each gene block is greater than 50 base pairs.
Dependencies are inferred from the claim text.