Abstract
BackgroundAlthough a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process.ResultsHere 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. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. 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. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. 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.ConclusionOur 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.
Highlights
A variety of methods and expensive kits are available, molecular cloning can be a timeconsuming and frustrating process
As a proof of principle, we subcloned several cDNAs into different vectors (human nicotinic receptor a4, a9 and b2 subunits, and serotonin receptor type 3A (5-HT3A) subunit into the pGEMHE vector, human endothelial cell-specific molecule 2 (ECSM2) into the p3XFLAG-CMV-14 vector, and Akt3v1 or Akt3v2 into pLXSN vector). pGEMHE is a vector containing 5’ and 3’ untranslated regions (5’UTR and 3’UTR) of Xenopus b-globin, which is highly expressed in Xenopus oocytes
We first optimized cloning conditions by cloning a9 nAChR subunit cDNA into the pGEMHE vector
Summary
A variety of methods and expensive kits are available, molecular cloning can be a timeconsuming and frustrating process. 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 [1]. This is a widely used method, it involves multiple steps and is time consuming. To overcome the difficulties encountered in the original cloning method, many other alternative cloning methods have been developed over the last two decades.
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