Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

TLDR

The largely unused uracil‑excision molecular cloning technique has excellent features compared to other modern cloning methods, but its application has been hampered by incompatibility with proof‑reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof‑reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique—simplicity, speed, high efficiency, and low cost—are now combined with high fidelity and a transparent, simple, flexible vector design, and a comprehensive set of vectors covering a wide range of applications has been constructed and their functionality confirmed.

Abstract

The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed.