Gene synthesis method

• US 6,110,668 A
• Filed: 12/20/1996
• Issued: 08/29/2000
• Est. Priority Date: 10/07/1996
• Status: Expired due to Fees

• Current Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Original Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Inventors: Schell, Jeff, Koncz, Csaba, Strizhov, Nicolai
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/769,802
• Time in Patent Office: 1,348 Days
• Field of Search: 435/6, 435/5, 435/91.1, 435/91.2, 435/4, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.16
• CPC Class Codes: C07K 14/325 Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
• 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 15/66 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
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

A method of gene synthesis is disclosed. The gene synthesis method permits the codons of [m] natural gene to be changed to allow preferential transcription and translation of the synthetic gene in transgenic organisms. The method utilizes a combination of enzymatic and chemical synthesis of DNA and significantly reduces the cost, time and number of steps required for construction of synthetic genes.

Claims

1. A method of constructing a synthetic single-stranded DNA molecule having an altered sequence relative to a template comprising:

(a) providing a single-stranded DNA template;

(b) chemically synthesizing a plurality of single-stranded oligonucleotides which are partially complementary to said template and form a contiguous sequence upon hybridization to said template, wherein mismatches between said oligonucleotides and said single-stranded template are located at any position internal to said oligonucleotides, and wherein the 5'"'"' end of any annealed oligonucleotide which is located at the junction point with another annealed oligonucleotide is phosphorylated; and

(c) without any PCR amplification of said oligonucleotides, subjecting a mixture comprising said template, said oligonucleotides, and thermostable DNA ligase to repeated cycles of increased and lowered temperature to achieve repeated melting and annealing of DNA strands, whereby a synthetic single-stranded molecule DNA strand is produced by ligation of adjacent oligonucleotides.

4. The method of claims 1, 2 or 3 wherein said oligonucleotides are produced by synthesis on a automated DNA synthesizer.

5. The method of claim 4 wherein said oligonucleotides which are phosphorylated at their 5'"'"' end are the result of a phosphorylation step on the DNA synthesizer, said phosphorylation occurring at the end of synthesis of said oligonucleotide.

6. The method of claims 1, 2 or 3 wherein said thermostable DNA ligase is Pfu.

2. A method of constructing a synthetic double-stranded DNA molecule having an altered sequence relative to a template comprising:

(a) providing a single-stranded DNA template;

(b) chemically synthesizing a plurality of single-stranded oligonucleotides which are partially complementary to said template and form a contiguous sequence upon hybridization to said template, wherein mismatches between said oligonucleotides and said single-stranded template are located at any position internal to said oligonucleotides, and wherein the 5'"'"' end of any annealed oligonucleotide which is located at the junction point with another annealed oligonucleotide is phosphorylated;

(c) without any PCR amplification of said oligonucleotides, subjecting a mixture comprising said template, said oligonucleotides, and thermostable DNA ligase to repeated cycles of increased and lowered temperature to achieve repeated melting and annealing of DNA strands, whereby a synthetic single-stranded molecule DNA strand is produced by ligation of adjacent oligonucleotides;

(d) producing an oligonucleotide primer complementary only to said synthetic DNA strand; and

(e) subjecting a mixture comprising said synthetic DNA strand, said primer, and thermostable DNA polymerase to repeated cycles of increased and lowered temperature to achieve repeated melting and annealing of DNA strands, whereby a double-stranded DNA molecule is produced.

8. The method of claim 2 or 3 wherein said synthetic double-stranded DNA molecule comprises a Bacillus thuringiensis toxin gene.

3. A method of constructing a synthetic double-stranded DNA molecule having an altered sequence relative to a template comprising:

(a) providing a single-stranded DNA template;

(b) chemically synthesizing a plurality of single-stranded oligonucleotides which are partially complementary to said template and form a contiguous sequence upon hybridization to said template, wherein mismatches between said oligonucleotides and said single-stranded template are located at any position internal to said oligonucleotides, and wherein the 5'"'"' end of any annealed oligonucleotide which is located at the junction point with another annealed oligonucleotide is phosphorylated;

(c) without any PCR amplification of said oligonucleotides, subjecting a mixture comprising said template, said oligonucleotides, and thermostable DNA ligase to repeated cycles of increased and lowered temperature to achieve repeated melting and annealing of DNA strands, whereby a synthetic single-stranded molecule DNA strand is produced by ligation of adjacent oligonucleotides;

(d) producing oligonucleotide primers which in PCR reactions only amplify the synthetic DNA strand; and

(e) subjecting a mixture comprising said synthetic DNA strand, said primers, and thermostable DNA polymerase to repeated cycles of increased and lowered temperature to achieve repeated melting and annealing of DNA strands, whereby a double-stranded DNA molecule is produced.

7. The method of claim 3 wherein at least one of said oligonucleotide primers for a PCR reaction is further designed to create a restriction enzyme site in the PCR product.