title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method.
abstract: BACKGROUND
  Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process.
  
  
  RESULTS
  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. 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.
  
  
  CONCLUSIONS
  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.
authors: Division of Neurobiology, Barrow Neurological Institute, St Joseph's Hospital and Medical Center, Phoenix, AZ, USA.
authors: Chaokun
authors: C
authors: Li
authors: Aiyun
authors: A
authors: Wen
authors: Benchang
authors: B
authors: Shen
authors: Jia
authors: J
authors: Lu
authors: Yao
authors: Y
authors: Huang
authors: Yongchang
authors: Y
authors: Chang
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli; D005822:Genetic Vectors / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014169:Transformation, Bacterial; D014982:Xenopus laevis
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes