A method of gene synthesis is disclosed

Gene synthesis is an efficient and cost-effective alternative to molecular cloning for custom gene production, where the DNA is manufactured by assembling strings of oligos together.Gene synthesis is the process of chemically synthesizing double-stranded DNA molecules in vitro. The main concept of gene synthesis is to assemble custom oligos into long DNA molecules. Email:marketing@medicilon.com.cn web:www.medicilon.com

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.

The present invention relates to a method of gene synthesis and more particularly to a gene synthesis method wherein the codons of a natural gene can be changed to allow optimal transcription and translation of the gene in transgenic organisms which prefer particular codons and other sequences that differ from those of the natural gene.

The degeneracy of the genetic code permits substantial freedom in the choice of codons for any particular amino acid sequence. Transgenic organisms such as plants frequently prefer particular codons which, though they encode the same protein, may differ from the codons in the organism from which the gene was derived. For example, U.S. Pat. No. 5,380,831 to Adang et al., relates to the creation of insect resistant transgenic plants that express the Bacillus thuringiensis (Bt) toxin gene. The Bt crystal protein, an insect toxin, is encoded by a full-length gene that is poorly expressed in transgenic plants. In order to improve expression in plants, a synthetic gene encoding the protein containing codons preferred in plants was substituted for the natural sequence. The invention disclosed therein comprised a chemically synthesized gene encoding an insecticidal protein which is frequently equivalent to a native insecticidal protein of Bt. The synthetic gene was designed to be expressed in plants at a level higher than a native Bt gene.

The approach used to assemble the Bt gene in the '831 patent is one of several methods for gene synthesis for genetic engineering manipulation. It consisted of designing an improved nucleotide sequence for the coding region and assembling the gene from a number of short chemically synthesized oligonucleotide segments. The DNA sequence to be synthesized was divided into segments, with lengths that can be synthesized, isolated and purified. The segments were then joined enzymatically to form the synthetic Bt gene.

Disadvantages of the gene synthesis method described in the '831 patent include its speed, costs, and efficiency. The approach is very sensitive to the secondary structure of oligonucleotides (e.g., hairpin loops) which interfere with the assembly. Hence, the approach has low efficiency and is not reliable for construction of long synthetic DNA fragments. Moreover, the method involves numerous steps and requires synthesis of both strands of the DNA. It requires purification of many short (50-70 nt) oligonucleotides by rather laborious and time consuming procedures. Use of T4 DNA ligase requires conventional temperatures (20°-37°C.) during assembly and ligation of oligonucleotides therefore contributing to the sensitivity of the method to secondary DNA structure.

Another approach to gene synthesis is described in U.S. Pat. No. 4,652,639 to Stabinsky. That reference also relates to a method for DNA synthesis wherein the synthetic gene has alternative codons selected on the basis of preferential expression in a projected host organism to be transformed. The method employed involves the ligation of two or more DNA strands although no template is employed. A key disadvantage of the gene synthesis method described in the '639 patent is that it can only be employed to synthesize short genes of about 200 base pairs. It is, however, frequently desirable to create longer genes.

Yet another method for the synthetic assembly of oligonucleotides into long DNA fragments utilizes polymerase to fill in single-stranded gaps in annealed pairs of oligonucleotides. However, after the polymerase reaction, each segment must be cloned, a step which significantly delays the synthesis of long DNA fragments and greatly decreases the efficiency of the approach. Additionally, the approach can be used only for small DNA fragments.

Recently, several PCR based techniques have been developed for construction of synthetic genes, where an assembly of overlapping oligonucleotides is performed by a thermostable DNA polymerase during repeated cycles by melting, anneling and polymerization. Although PCR mediated methods are rather simple and labor saving, they are not free from drawbacks. A key disadvantage of assembly by PCR is that complex mispriming events negatively exert the correctness of a resulting assembled DNA. In addition, the low fidelity of thermostable DNA polymerase influences the reliability of this technology with increased number of PCR steps.