co9b00167_si_001.pdf (444.66 kB)
In Vitro Construction of Large-scale DNA Libraries from Fragments Containing Random Regions using Deoxyinosine-containing Oligonucleotides and Endonuclease V
journal contribution
posted on 2020-03-26, 13:39 authored by Yasuhide Yamamoto, Takuya Terai, Shigefumi Kumachi, Naoto NemotoEfficient
and precise construction of DNA libraries is a fundamental starting
point for directed evolution of polypeptides. Recently, several in
vitro selection methods have been reported that do not rely on cells
for protein expression, where peptide libraries in the order of 1013 species are used for in vitro affinity selection. To maximize
their potential, simple yet versatile construction of DNA libraries
from several fragments containing random regions without bacterial
transformation is essential. To address this issue, we herein propose
a novel DNA construction methodology based on the use of polymerase
chain reaction (PCR) primers containing a single deoxyinosine (I)
residue near their 5′ end. Treatment of the PCR products with
endonuclease V generates 3′ overhangs with customized lengths
and sequences, which can be ligated accurately and efficiently with
other fragments having exactly complementary overhangs. As a proof
of concept, we constructed an artificial gene library of single-domain
antibodies from four DNA fragments.