Figure 1.
Schematic outline of inverse fusion PCR cloning (IFPC). (Primer design)
3 primers are required for IFPC. For the amplification of the insert, the forward primer A and the reverse primer B are used. Primer B is an insert-specific standard primer while the 5′-end of primer A is comprised of a sequence homologous to the desired insertion site of the vector (black) and the 3′-end is specific for the insert (white). The annealing site for the vector primer C has to be chosen downstream of the insertion site, and must not overlap with the insertion site. The annealing Tm of primer B, primer C, the vector homologous part of primer A as well as the insert specific part of primer A should all be around 58°C. Depending on how IFPC will be performed, primer B or primer C can be 5′-phosphorylated (see below). The sequence between the insertion site and primer C will be deleted after IFPC. (Inverse fusion PCR cloning) (1.) The insert (white) is amplified via primers A and B and should be gel-eluted when unspecific PCR products or smears appear. (2.) For the inverse fusion PCR, a mix containing insert-PCR product, circular plasmid template, primer B and phosphorylated primer C is prepared. In the first rounds of PCR, forward strands of vector and reverse strands of insert are enriched by primer-extension of primers B and C in a linear way (2.1). Then, the insert reverse strands anneal with their vector homologous 3′-end to the complementary sequence (black) of the linear plasmid forward strands (2.2.) and the inserts are elongated by overlap extension (2.3.), thus forming the fused insert-plasmid template lacking the original sequence of the template plasmid between the insertion site and primer C (2.4.). The second strand of the template is generated by primer extension of primer C, finalizing the double-stranded template (2.5.), which is now exponentially amplified via primer B and C (2.6.). The linear insert-plasmid fusions are now circularized by T4-ligation (3.). As an alternative to phosphorylated primer C, a phosphorylated primer B can be used, or the phosphorylation can be incorporated by T4-polynucleotide-kinase treatment during the ligation step. Finally the ligated insert-vector fusions are transformed into competent E.coli (4.), where the bacterial DNA repair machinery will close the nick of the second strand. A working protocol is shown in material and methods.
Figure 2.
Experimental setup for the exchange of antibiotic resistances by inverse fusion PCR cloning.
(A) The ampicillin resistance gene of the vector pBAD was exchanged by a kanamycin resistance gene. Via selection on ampicillin plates the residual background was detected, and through kanamycin selection the functional fusions were identified. This setup positively selected functional fusions and was used to optimize the system. (B) The kanamycin gene of the vector pCR2.1 was exchanged by an in-frame insertion of a spectinomycin gene (aadA). (1) In a first round, the ampicillin resistant colonies were selected, containing the vector with or without insertion. (2) In a second round, 192 of the ampicillin resistant colonies were used. Background containing the original vector was selected in LB media by addition of kanamycin and functional spectinomycin fusions by addition of spectinomycin. (3) 10 spectinomycin resistant clones were sequenced for sequence confirmation and all clones sensitive to kanamycin and spectinomycin were sequenced to identify the reason of IFPC failure. With this setup the insertion of non selectable sequences could be mimicked, because background (kanR), successful insertions (specR) and failed fusions (kanS/specS) were detected. By calculating the relationship between successful insertions and failed fusions, the failure frequency of IFPC was specified to be 6.5% in this experiment. (C) Results of an inverse fusion PCR to change the ampicillin into a kanamycin resistance in the vectors pBAD (lanes 1–7) or pBAD-TOPO/lacZ/V5-His (lanes 8–10). The molarity of vector and insert templates in lanes 1–7 correspond to the amounts shown in Table 1 (N°. 1–7) and in lanes 8–10 to the amount shown in Table 1 (N°. 7), but 25 cycles (lane 8), 30 cycles (lane 9) and 35 cycles (lane 10), respectively, were performed in the inverse fusion PCR step. The PCR was run using primer B1 and primer C1-b. 10 µl of each PCR were loaded on the gel and resulted after ligation and transformation in 260.000 cfu (lane 7), 12.000 cfu (lane 8), 19.600 cfu (lane 9) and 15.700 cfu (lane 10) per µl of inverse fusion PCR. M: Bench Top1 kb ladder (Promega).
Table 1.
Conditions and cloning rates for kanamycin insertion into pBAD by IFPCa.
Table 2.
Oligonucleotides.