Page 257 - Advances in Textile Biotechnology

238 Advances in textile biotechnology

2 Random method or ‘concatemerization’, where an uncontrolled number
of monomer DNA segments are oligomerized in a single step, creating
a population of oligomerized clones of different lengths. This method
has the advantage of creating, in a simple ligation step, a library of genes
of different lengths that encode oligomeric polypeptides with the same
repeat sequence. However, it sacrifices precise control over the oligo-

merization process, because it is a statistical process and, therefore, it is
diffi cult to, a priori, design a polypeptide of a certain size and diffi cult
to synthesize genes encoding large polypeptides (>100 kDa).
3 Recursive directional ligation (RDL), where DNA segments with two
different restriction sites flanking the insert are joined in sequential

steps with the length of the ligated segments growing geometrically in
each step. This approach is applicable for the synthesis of repetitive
polypeptides of specific and predetermined chain length because this

process seamlessly joins the two monomeric inserts and also eliminates
the restriction site at the junction, so that the two unique restriction sites
are now on either end of the dimerized gene (Fig. 10.2) (Chilkoti et al.,
2002, Rodríguez-Cabello et al., 2008).

E E
E E E E E

E E E
Iterative
E
E
E
E E E
E E
E
E
Random

E E
E E E E E

E E E
Recursive
10.2 Schematic representation of three DNA oligomerization
techniques (adapted from Rodríguez-Cabello et al., 2008 and Chilkoti
et al., 2002). ‘E’ represents the targets for restriction enzymes fl anking
the inserts that are joined in the construction of the repetitive
polypeptides.

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