LONG NUCLEIC ACID SEQUENCES CONTAINING VARIABLE REGIONS

• US 20150159152A1
• Filed: 12/09/2014
• Published: 06/11/2015
• Est. Priority Date: 12/09/2013
• Status: Abandoned Application

• Current Assignee: Integrated DNA Technologies, Inc. (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies, Inc. (Danaher Corporation)
• Inventors: Allen, Shawn, Rose, Scott, Beltz, Kristin
• Application Number:
  US14/564,504
• Publication Number:
  US 20150159152A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

This invention pertains to improved methods for the synthesis of long, double stranded nucleic acid sequences containing difficult to clone or variable regions.

Claims

1. 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.

2. The method of claim 1 wherein the first gene block is greater than 50 base pairs and the second gene block is greater than 50 base pairs.

3. The method of claim 1 wherein the mixture further comprises one or more additional gene blocks wherein the one or more bridging oligonucleotides contain one or more regions that are hybridizable to a portion of the one or more additional gene blocks.

4. The method of claim 1 wherein the mixture further comprises one or more additional gene blocks and one or more additional bridging oligonucleotides wherein the one or more additional bridging oligonucleotides contains (i) a region hybridizable to an additional gene block, and (ii) a region hybridizable to another additional gene block, the first gene block or the second gene block.

5. The method of claim 1 wherein the mixture is assembled and amplified less than twenty PCR cycles.

6. The method of claim 1 wherein the mixture is assembled and amplified between 5 and 15 PCR cycles.

7. The method of claim 1 wherein the bridging oligonucleotide set is comprised of bridging oligonucleotides containing at least one degenerate base.

8. The method of claim 1 wherein the bridging oligonucleotide set is comprised of bridging oligonucleotides containing from 1-30 degenerate bases.

9. The method of claim 1 wherein the bridging oligonucleotide set contains at least one mismatch or non-standard base located within the first region or second region.

10. The method of claim 1 wherein the bridging oligonucleotide set contains fixed regions of low complexity, direct or indirect repeats, and/or homopolymeric nucleotide runs.

11. The method of claim 1 wherein the bridging oligonucleotide set consists of a sequence that is hybridizable to the first gene block and sequence that is hybridizable to a second gene block, and upon assembly does not add an additional sequence between the first and second gene blocks.

12. The method of claim 1 wherein the bridging oligonucleotide set is comprised of bridging oligonucleotides wherein the first hybridizable region is between 10-50 bases and the second hybridizable region is between 10-50 bases.

13. The method of claim 1 wherein the bridging oligonucleotide set comprises two or more bridging oligonucleotides with an identical sequence except for mixed base site locations varying along the bridge sequence of the bridging oligonucleotide(s) that did not hybridize to a gene block.

14. The method of claim 1 wherein the bridging oligonucleotide set contains non-random nucleotide variation at specific location(s).

15. The method of claim 14 wherein the non-random variation at specific locations is for targeted codon changes.

16. The method of claim 1 wherein the bridging oligonucleotide set contains a region of low complexity or repeating elements.

17. The method of claim 1 wherein the mixed base molar ratios in a variable region of a bridging oligonucleotide set is controlled by hand mixing phosphoramidites at the desired ratio.

18. 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.

19. 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.

21. The kit of claim 19 further comprising multiple bridging oligonucleotides containing varying regions of degenerate bases.

20. The kit of claim 20 wherein each gene block is greater than 50 base pairs.