![]() However, it includes two sequential 35-cycle PCRs with a total number of 70 cycles. One-step "quick assemble" cloning does not need purification of PCR products. Similarly, overlap extension PCR cloning also requires purification of the first round PCR products (vector and insert) and an additional round overlap extension PCR, which usually generates multiple bands, for producing linked vector and insert. However, the new ligation independent cloning still requires purification of the digested vector and PCR-amplified insert, and the purchase of purification and cloning kits. The latest ligation-independent cloning, such as CloneEZ and In-Fusion cloning kits, uses some DNA polymerase to generate sticky ends in the vector and insert without specific sequence requirement, except for restriction sites to linearize the vector. Gateway recombinational cloning uses site-specific recombination to transfer cDNAs between donor and destination vectors, which requires additional specific sequences for recombination. This technique requires specific sequences to create 15-base overhangs. The early ligation independent cloning uses the 3'-exonucnease activity of T4 DNA polymerase to create 15-base 5'overhangs in the ends of insert and complementary 5' overhangs in the ends of vector. The limitations of this method are low fidelity of Taq DNA polymerase causing unwanted mutations and requirement of subcloning into the final target vector with restriction digestion and ligation. The PCR product is directly cloned into a TA cloning vector with a complementary 3'-T overhang in both ends without restriction digestion. For example, TA cloning uses regular Taq DNA polymerase to add a single 3'-A overhang to the ends of the PCR product. However, each of these techniques has its own limitations. These methods include TA cloning, ligation independent cloning with T4 DNA polymerase, GATEWAY recombinational cloning, and more recent sequence- and ligation-independent cloning kits, such as CloneEZ (GenScript USA Inc., Piscataway, NJ, USA), one step cloning, and overlap extension PCR cloning. To overcome the difficulties encountered in the original cloning method, many other alternative cloning methods have been developed over the last two decades. This multi-step process also makes it difficult or complicated for troubleshooting. Although this is a widely used method, it involves multiple steps and is time consuming. Traditionally, molecular cloning joins insert and vector by T4 DNA ligase after restriction digestion to excise insert from a donor vector or from a PCR product with restriction enzyme recognition sites added to the ends. Molecular cloning is one of the most widely used techniques in biomedical research laboratories. Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. In addition, this method is highly effective and reproducible. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. Second, there is no need of any cloning kit or specialized enzyme for cloning. First, it does not need gel purification of the PCR product or linearized vector. This technique has many advantages over other cloning methods. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA) in a vector at any desired position. Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process.
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