The desired gene coding for an artificial protein is divided into a series of 5´-end overlapping complementary oligonucleotides. To achieve maximal benefit from the gene assembly, the sequence of the oligonucleotides to be assembled has to be planned carefully with the following considerations:
- The formation of hairpins of 4 or more bases should be avoided.
- Sequences that are commonly associated with poor coupling efficiency during the chemical synthesis should be avoided.
- It is recommended to avoid sequences that introduce rare codons into a gene, if you want to achieve high levels of gene expression (protein production).
- The generation of unique restriction sites within the assembled gene will allow subsequent manipulations by recombinant DNA techniques.
Eurofins Genomics uses their proprietary software GENEius for the design of the oligonucleotides.