Synthesis of Total sFat-1 Gene by PCR-Restrict Enzyme Ligation Method

Abstract

Gene synthesis is very important in life science research, and it becomes a technique in common use. It is difficult for long gene synthesis, because the mismatches and mutations of DNA sequence in nucleotide fragments assembling. This study established a new method for long gene synthesis, which was referred to as PCR-restrict enzyme ligation method. With this method, a omega-3 fatty acid desaturase gene, sFat-1, from Caenorhabditis briggssae, was successfully assembled from 27 synthesized nucleotide fragments (60 ~ 68 bp for each fragment ) following 3 rounds of PCR (7 reactions) and 2 rounds of restrict enzyme ligation (3 reactions). This shows that the PCR-restrict enzyme ligation method is an effective method for long gene synthesis.