Analysis of Assembly of Synthetic Antibody Fragments: Expression of Functional scFv with Predefined Specificity

In this work, we have investigated parameters important for the assembly of synthetic genes encoding antibody fragments. These genes are constructed from a set of overlapping single-stranded oligonucleotides (primers), which are assembled into a gene sequence in a one-step process using PCR. Using the Oligo program, we made a detailed analysis of wanted and unwanted interactions between these primers; both the stability of hairpin structures of homodimers and of heterodimers were examined. The Oligo program could be used to identify unwanted interactions of high stability, and the present study suggests that if the stabilities of the unwanted interactions are kept to 25%-50% of the designed interactions, a successful assembly of synthetic genes can occur.