The In-Fusion HD Cloning Kit, developed by Takara Bio, is an efficient, seamless DNA cloning technology used in molecular biology laboratories. This method offers a streamlined way to insert one or more DNA fragments into a linearized vector, bypassing the need for traditional restriction enzymes or DNA ligase. The technology uses a proprietary enzyme that fuses DNA fragments together by recognizing short, complementary sequences engineered onto their ends. This technique is a modern standard for constructing complex DNA plasmids with speed and high fidelity.
The Enzymatic Mechanism of Fusion
The core of the In-Fusion system is a proprietary enzyme that facilitates the precise joining of DNA fragments. This enzyme recognizes short stretches of homologous sequence (typically 15 to 20 base pairs) at the ends of the linearized vector and the DNA insert. The reaction works by exploiting the enzyme’s 3’ to 5’ exonuclease activity in the absence of deoxynucleotide triphosphates (dNTPs).
When the linearized vector and the insert fragments are mixed with the In-Fusion enzyme, the exonuclease activity begins to “chew back” the 3’ ends of the double-stranded DNA. This results in single-stranded 5’ overhangs on both the vector and the insert. These overhangs are complementary to each other due to the pre-designed homologous sequences.
The single-stranded overhangs then anneal to one another, effectively fusing the DNA fragments together in the correct orientation. This annealing creates a circular DNA molecule containing nicks and gaps. Upon transformation into competent Escherichia coli cells, the bacterial host’s DNA repair machinery naturally seals these remaining nicks and gaps. This results in a covalently closed, functional recombinant plasmid.
Designing Homologous Overlaps
Successfully using the In-Fusion method depends on the precise design of the DNA fragments to ensure homologous overlaps are present. Both the vector and the insert must share a short, identical sequence at each junction. The standard recommendation is an overlap of 15 base pairs, though increasing this to 20 base pairs often improves cloning efficiency, especially when assembling multiple fragments.
The insert fragment is typically prepared by Polymerase Chain Reaction (PCR) amplification. To create the required homology, the PCR primers must be designed with two distinct regions: a 3’ end specific to the target gene, and a 5’ end that acts as a tail containing the 15-20 bp sequence identical to the end of the linearized vector. This design ensures that the final amplified insert carries the vector’s terminal sequence on its ends.
The gene-specific portion of the primer, usually 18–25 nucleotides in length, should follow standard primer design rules, including a melting temperature (Tm) between 58°C and 65°C to ensure efficient PCR amplification. The 5’ homologous tail sequence should not be included in the Tm calculation, as it does not bind to the template DNA during the initial annealing steps of PCR.
Key Benefits for Cloning Projects
The In-Fusion system offers several advantages over older molecular cloning techniques due to its sequence-independent nature. Unlike traditional methods that rely on specific restriction enzyme recognition sites, In-Fusion allows any insert to be cloned into any location within a vector, providing flexibility in construct design. The resulting construct is seamless, meaning the assembled DNA contains no extra bases or “scar” sequences at the junction points, which is desirable for creating fusion proteins or maintaining a precise reading frame.
Another benefit is the inherent directionality of the cloning, as the unique homologous overlaps ensure the insert is incorporated in only one intended orientation. This eliminates the need for screening clones that have the insert in the reverse orientation, a common issue with blunt-end ligation. The technology also excels in multi-fragment assembly, allowing researchers to join up to five or more DNA fragments, plus the vector, in a single, one-step reaction, which speeds up complex cloning projects.
The speed and simplicity of the workflow contribute to its utility in high-throughput settings. The reaction requires only a single, short incubation step. Furthermore, the high cloning accuracy—often exceeding 95% correct colonies—minimizes the time spent on downstream colony screening and sequencing.
Overview of the Laboratory Protocol
The laboratory procedure for In-Fusion cloning begins with the preparation of the vector and insert DNA fragments. The vector must first be linearized at the intended insertion site, which can be accomplished using a restriction enzyme digest or by inverse PCR. The linearized vector must be purified to remove any remaining circular plasmid DNA that could lead to high background colonies.
Concurrently, the insert DNA fragments are generated using PCR with the specially designed primers that incorporate the 15–20 bp homologous tails. Following amplification, the PCR products must be purified, typically through spin-column purification or gel extraction, to ensure high quality and concentration. In cases where the PCR product is clean, a proprietary Cloning Enhancer can be used to treat the product, bypassing the purification step.
The fusion reaction involves mixing the linearized vector, the purified insert(s), and the In-Fusion HD Enzyme Premix. This mixture is then incubated for a short period, commonly 15 minutes, at a temperature of 50°C. This incubation allows the enzyme to perform the exonuclease activity and enables the complementary ends to anneal.
Finally, the reaction mixture is used to transform chemically competent E. coli cells. The transformation is followed by a brief recovery period in a nutrient medium before the cells are plated onto selective agar plates containing the appropriate antibiotic. The host cell’s repair mechanisms complete the circularization of the recombinant plasmid, and colonies containing the successfully cloned construct grow overnight, ready for subsequent analysis.