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

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.

Figure 1

Overview of the USER cloning technique. A PacI cassette containing USER vector (upper left corner) is digested with PacI and Nt.BbvCI to generate 8 nt single-stranded 3′ overhangs. A PCR fragment amplified with compatible uracil-containing primers by the PfuTurbo^® C_x Hotstart DNA polymerase is mixed with USER™ enzyme mix (removing uracils, pink) and the linearized vector. The mixture is incubated 20 min at 37°C and 20 min at 25°C, and the hybridized product is ready to be transformed into E.coli without prior ligation. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark single base differences between the generated 3′ overhangs, which are responsible for the directional insertion of the PCR fragment.

Figure 2

Characteristics and applications of the USER technique. (a) A comparison of the ability of DNA polymerases to amplify DNA fragments using uracil-free (-dU) and uracil-containing primers (+dU). Hm Taq: HotMaster™ Taq DNA Polymerase, Taq: Platinum^® Taq DNA Polymerase, Pwo: Pwo DNA Polymerase, Phusion: Phusion™ DNA Polymerase, PfuCx: PfuTurbo^® C_x Hotstart DNA polymerase. (b) Functional expression of A.thaliana glucose transporter AtSTP1 in X.laevis oocytes from the Xenopus-specific USER compatible vector, pNB1u. H₂O: control oocytes injected with water; STP1: oocytes injected with AtSTP1 cRNA. (c) Functional expression of the cyano fluorescence protein in leaf and root of A.thaliana from the CaMV 35S promoter using the USER compatible pCAMBIA230035Su vector (35S:CFP). Control: empty pCAMBIA230035Su.

Figure 3

Sequential USER cloning of multiple inserts. Inclusion of 25 bp of the PacI cassette sequence in the reverse primer used to amplify a DNA fragment prior to USER cloning results in regeneration of the PacI cassette downstream of the inserted fragment. For smaller fragments the entire insert can be assembled from chemically synthesized oligonucleotides. Subsequent digestion of the construct with PacI and Nt.BbvCI allows insertion of another fragment into the vector by USER cloning. Sequentially inserted DNA fragments will have a minimum of 13 bp sequence between them. Nt.BbvCI recognition sites are marked in tan, PacI recognition sites are marked in light blue. Yellow and green mark the single base differences between the generated 3′ overhangs.