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Figure 2 | BMC Molecular Biology

Figure 2

From: Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

Figure 2

The USER Friendly cloning strategy for single step construction of replacement vectors. A) Amplification of the two homologous recombination sequences (HRS) with primers that contain 5' deoxyuridine extensions. B) Treatment of the PCR amplicons with USER enzyme mix, resulting in the generation of unique 3' single stranded overhangs. The USER enzyme solution is a mixture of Uracil DNA glycosylase and DNA glycosylase-lyase Endo VIII. The Uracil DNA glycosylase recognises the 2'-Deoxyuridine base in the primer portion of the PCR amplicon and excises the uracil nucleobase, resulting in an abasic position [30]. The presence of an abasic site in the DNA permit the DNA glycosylase-lyase Endo VIII to break the phosphodiester backbone at both the 3' and 5' sides of the abasic position, resulting in a single strand break [31]. The resulting short 5' stretch of the original primer then dissociates, leaving the PCR fragment with a 9 bp long 3' single stranded overhang. C) Design of the USER vector for targeted gene replacement in fungi, with two unique USER cloning sites (LB and RB). Each of the UCS's consists of a PacI site (Red), two Nt.BbvCI sites (blue) and two times two unique base pairs (yellow, green, gray and pink) ensuring directional cloning of the inserts. Digestion of the vector results in the generation of two DNA fragments with four unique 9 bp long 3' overhangs. D) Mixing and annealing of the two vector DNA fragments and the two inserts. The four unique 3' overhangs ensures correct annealing between the four DNA fragments. E) Transformation into E. coli, where covalent bonds are formed between the base-paired DNA fragments. F) Screening for correct transformants by colony-PCR using the HRS specific primer pairs that were used in step A. The figures are not drawn to scale.

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