In spite of many advantages, artificial genes for cytokines have seldom been prepared due to the difficulty of constructing long genes from short DNA oligomers. To improve the method for the construction of a cytokine gene, a new way to construct a long gene easily and rapidly was developed. Using a plus gene and a minus gene both as template and primer that have 20-40 bases of cohesive ends, a double stranded DNA is synthesized by the reaction of polymerase. We named that as the "SPR method".
First, the gene sequences of equine interferon alpha-1 and gamma were designed with consideration of the creation of a restriction site and the adaptation to yeast codon usage. Then, 8-11 of the DNA short oligomers (each is 80 mer-93 mer) containing plus genes and minus genes, were synthesized by a DNA synthesizer. Each set of plus and minus genes was mixed and reacted with rTaq DNA polymerase. Then, similar reactions were repeated with the sets of these products. The final product was about 600 bp in length, which indicated that the full-length gene should be prepared. As the result of DNA sequencing, it was shown that this artificial gene had a complete sequence of equine interferon alpha-1 and gamma.
Within only a week, about 600 bp of the artificial genes were easily, rapidly and completely constructed. More than 1,000 bp of gene or genome can be prepared using the SPR method. (Lab. of Epizootiology, Department of Exotic Diseases TEL +81-42-321-1441)
References:
CYTOKINE, 11 : p.927 (1999)
