Optimized in-Fusion Cloning System Demonstrates High Efficiency and Accuracy of Multi-Fragment Cloning

Optimized In-Fusion cloning system demonstrates high efficiency and accuracy of multi-fragment cloning
Steve Oh1, Gia Jokhadze1, Tommy Duong1, Magnolia Bostick1, Andrew A. Farmer1

1Clontech Laboratories, Inc., Mountain View, CA

In-Fusion PCR Cloning technology enables directional, seamless cloning of any PCR fragment into any linearized vector in a single 15-minute reaction. No additional treatment of the PCR fragment is required (such as restriction digestion, ligation, phosphorylation, or blunt-end polishing). In contrast, the In-Fusion enzyme generates short regions of single-strand overlap between vector and insert, so as to drive directional cloning of the desired fragment. This overlap can be designed into the PCR primers which amplify the desired sequences. While cloning single inserts is extremely efficient, cloning experiments are becoming increasingly complex, involving multiple fragments to stitch genes together from synthetic building blocks or to engineer new functionalities through combinatorial protein domain swapping, etc. This, in some cases, may present a challenging task. Here, we present data showing multi-fragment cloning efficiency and accuracy can be significantly increased by optimizing various parameters of the In-Fusion Cloning reaction. This extends the versatility of the In-Fusion cloning system to meet the coming challenges of innovative research in the synthetic biology field.