Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes (original) (raw)
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A Rapid and Simple, Recombination-Based Cloning Method in Escherichia coli
BIOSCIENCES BIOTECHNOLOGY RESEARCH ASIA, 2017
Cloning is indispensable in molecular biology. Here we developed an in vivo homologous recombination-based cloning procedure and determined the optimal conditions. This procedure required two PCR products to be amplified from a gene of interest and desired plasmid vector. The 5' ends of both primers that amplified one product had nucleotide sequences complementary to that used to amplify the other product. Once the mixture of those PCR products was introduced into Escherichia coli DH5α competent cells, transformants carried plasmids in which the gene of interest had been properly cloned. Optimizing the cloning conditions, at least 12-nucleotides overlaps between the terminal ends of two fragments were required to generate desired plasmids. This value was much shorter than the length of overlaps required for the same procedure employed in the yeast system. Therefore, this procedure is expected to be an attractive alternative for cloning in the E. coli system. Gene cloning is an indispensable tool in molecular biology. Conventional cloning utilizes restriction endonucleases to generate DNA fragments with complementary ends and a DNA ligase to connect these fragments prior to transformation. However, this process requires multiple steps and is time consuming. Therefore, many previous attempts have been made to overcome the limitations of the conventional cloning, e.g., the substitution of restriction endonucleases with T4 DNA polymerase or exonuclease III to generate DNA fragments [1,2] , ligation-independent cloning [3,4] , enzyme-free cloning [5] , overlap extension PCR cloning [6] ,
Applied and Environmental Microbiology, 1998
An extremely thermostable restriction endonuclease,PspGI, was purified from Pyrococcus sp. strain GI-H. PspGI is an isoschizomer of EcoRII and cleaves DNA before the first C in the sequence 5′ ^CCWGG 3′ (W is A or T). PspGI digestion can be carried out at 65 to 85°C. To express PspGI at high levels, the PspGI restriction-modification genes (pspGIR andpspGIM) were cloned in Escherichia coli. M.PspGI contains the conserved sequence motifs of α-aminomethyltransferases; therefore, it must be an N4-cytosine methylase. M.PspGI shows 53% similarity to (44% identity with) its isoschizomer, M.MvaI fromMicrococcus variabilis. In a segment of 87 amino acid residues, PspGI shows significant sequence similarity toEcoRII and to regions of SsoII andStyD4I which have a closely related recognition sequence (5′ ^CCNGG 3′). PspGI was expressed in E. coli via a T7 expression system. Recombinant PspGI was purified to near homogeneity and had a half-life of 2 h at 95°C. PspGI remained active following 30...
Applied and Environmental Microbiology, 2007
We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.
2017
The precise assembly of defined DNA sequences into plasmids is an essential task in bioscience research. While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning techniques based on restriction digestion and ligation are still commonly used in routine DNA cloning. Here, we describe a simple, fast, and economical cloning method based on RecA- and RecET-independent in vivo recombination of DNA fragments with overlapping ends using E. coli. All DNA fragments were prepared by a 2-consecutive PCR procedure with Q5 DNA polymerase and used directly for transformation resulting in 95% cloning accuracy and zero background from parental template plasmids. Quantitative relationships were established between cloning efficiency and three factors–the length of overlapping nucleotides, the number of DNA fragments, and the size of target plasmids–which can provi...