Introduction

DNA constructs are typically joined by ligation at restriction enzyme sites and construct options are limited by the available unique sites in the vector and gene. In contrast, an In-Fusion™ enzyme reaction can join any two pieces of DNA that have 15 bp of identity at their ends. The 15-bp overlap may be engineered by inclusion in primers used to PCR amplify a segment of DNA. The pieces of DNA may be generated by PCR and have blunt ends or by restriction digest of plasmid DNA and have sticky or blunt ends depending on the enzyme used. The In-Fusion mechanism is ligation-independent and while proprietary, likely uses the unique properties of the 3′–5′ exonuclease activity of poxvirus DNA polymerase (1,2,3). When incubated with linear duplex DNAs with homologous ends in the presence of Mg²⁺ and low concentrations of dNTP, the 3′–5′ proofreading activity of poxvirus DNA polymerase progressively removes nucleotides from the 3′ end. This exposes complementary regions on substrate DNAs that can then spontaneously anneal through base pairing, resulting in joined molecules containing a hybrid region flanked by nicks, 1–5 nucleotide gaps, or short overhangs (Figure 1A). The annealed structures are metastable because the poxvirus DNA polymerase has a lower affinity for nicked or gapped DNA ends than for duplex ends. Introduction into Escherichia coli repairs any single-stranded gaps. Thus, one copy of the overlap is present in the final DNA product, and the result is equivalent to a recombination event at the ends of the DNAs. Originally described for inserting one piece of DNA into a restriction enzyme-digested plasmid, we have found that In-Fusion can join four or more pieces of DNA. We used this insight to develop seamless fusion proteins, modular vectors, and novel mutagenesis strategies.

Figure 1. Mechanism of an In-Fusion reaction and its use in constructing a three-piece fusion protein. (Click to enlarge)

Materials and Methods

Design of a construct. The desired pieces of a DNA construct are assembled in a DNA manipulation program such as Sequencher™ (Gene Codes, Ann Arbor, MI, USA). For example, as shown in Figure 1B, DNA segments encoding the interleukin-2 (IL-2) signal sequence (4,5), the extra-cellular domain of CD101 minus its endogenous signal sequence (6), and the fragment crystallizable (Fc) domain of murine immunoglobulin G3 (IgG3) (7) are assembled with a mammalian expression vector.

Design of overlap primers. Sense and antisense PCR primers are designed, which contain a 15-bp overlap with the adjacent segment of the construct and 20–30 bp of segment-specific sequence. The junction between two pieces of DNA can be made seamless by including no additional DNA sequence. Alternatively, short pieces of DNA such as restriction sites, translation initiation sites, linkers, or epitope tags can be added by inclusion in the primer sequence. Table 1 and Table 3 give the primers for the constructs in Figure 1B and Figure 3, A and B. Vector segments can be generated by restriction enzyme digest of a plasmid or by PCR. Where a primer is designed to overlap a restriction-digested DNA fragment, the 15-bp overlap is counted from the cleavage site on the antisense DNA strand as described in the Clontech In-Fusion user manual. Most primers are 35-55 bp, and we find quality control by mass spectroscopy to provide sufficient purity (Midland Certified, Midland, TX, USA).

Figure 2. Plasmid minipreps of an In-Fusion reaction. (Click to enlarge)

Table 1. Primers for PCR Amplification of IL-2 Signal, CD101, and Murine IgG3 Segments (Click to enlarge)

Generation of DNA segments. The DNA segments were PCR amplified from appropriate templates with overlap primers (designed as described in the section entitled Design of overlap primers) and PfuUltra® II Fusion Hot Start polymerase (Stratagene, La Jolla, CA, USA), gel-purified, and quantitated. The use of a high-fidelity polymerase reduces errors, but Taq polymerase PCR products will also work.

In-Fusion reaction. Twenty-five to one hundred nanograms of restriction enzyme-digested, gel-purified vector were mixed at a molar ratio of 1 vector to 2 of each DNA segment in a total of 10 µL water in one tube of In-Fusion Dry-Down reaction mix (Clontech, Mountain View, CA, USA). The reaction was incubated at 42°C for 30 min, transferred to ice, and 40 µL Tris EDTA (TE) were added. Four microliters were transformed into One Shot® TOP10 competent E. coli (1 × 10⁹ cfu/µg; Invitrogen, Carlsbad, CA, USA), miniprepped, and characterized by restriction enzyme digest and sequencing.