e-book PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering

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Cohen initiated the genetic engineering era. PCR has been indispensible for gene cloning because it can provide target gene fragments in vitro in large amount and short time without the limitation of restriction sites. As a result, the mainstream strategy for the construction of recombinant expression vector includes two cloning steps: PCR cloning cloning of PCR amplified target gene into cloning vector and restriction enzyme mediated subcloning of target gene from cloning vector into expression vector. Direct cloning of PCR amplified target gene into expression vector via double-restriction of PCR products is optional, but the uncertainty of the accuracy of artificially synthesized restriction site in PCR primers makes trouble-shooting difficult if the cloning experiment fails.

T-A cloning Marchuk et al. Blunt-end PCR cloning kit is also available, but it has in fact the same drawback like T-A cloning because both of them are ligase-dependent. Restriction enzyme mediated subcloning is more time-and-money consuming because two times of double-restriction and gel purification are needed. Many ligase-independent PCR cloning strategies have been developed in order to overcome the inefficiency of ligase dependent PCR cloning, such as ligation independent cloning Aslanidis and Dejong ; Rashtchian et al.

In this cloning strategy, PCR amplified target gene is inserted into plasmid vector by means of a lineal recombination PCR using circular vector as template and target gene PCR products as primers, which contains two primer-introduced terminal sequences homologous to correspondent sequences in the vector. Although the PCRRC strategy in these two reports is more straightforward, efficient and reliable compared with other PCR-mediated cloning strategies, a restriction enzyme Dpn I digestion step is still necessary to eliminate template vector as selection for recombinant plasmids.

In order to overcome above mentioned inconvenience of constructing recombinant expression vector of a target gene, we elaborately designed and constructed an omnipotent expression vector designated as pOmni that has two advantageous technical features. All E. All mutational primers and regular primers were designed with Primer Premier 5.

Shanghai, China. All PCR products were visualized through agarose-gel electrophoresis if necessary. Transformation of Escherichia coli in all cloning experiment was conducted according to the standard chemical transformation protocol Sambrook and Russell Intermediate vector pC3. The destination vector pOmni was constructed based on pC3.


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The quantity of clones of individual cloning was count, and then 10 clones from individual cloning were randomly picked and regular colony PCR with correspondent primer set was conducted to confirm positive clones. The designed vector pOmni Fig.

Through two times of insertion mutation, the Lac operator and ribosome biding site RBS was introduced downstream T7 promoter originally used for sequencing in pcDNA 3. Physical map of pOmni. Suicide gene expression cassette of lysis gene E of bacteriophage Phi-X is to be replaced by cloned gene in PCRRC strategy so as to serve as positive selection gene for recombinant vector.

Detailed sequence information of designed functional elements in pOmni. PCR amplified target gene products containing two primer-introduced terminal sequences shaded sequences can be directionally and seamlessly inserted into correspondent position and replace the suicide gene through recombination PCR.

The designed Kozak sequence and its proper position related to RBS make it possible to express cloned target gene in both E. A series of TA-based and zero-background vectors for plant functional genomics.

Cloning - PCR Strategy - Primer Design - EMBL

Gabant, P. Engler, C. Generation of families of construct variants using golden gate shuffling. Golden Gate cloning. J Mol Biol , — Green, M. Molecular Cloning: A Laboratory Manual. Yanisch-Perron, C.

Background

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33 , — Positive-selection vectors using the F plasmid ccdB killer gene. Gene , 71—74 Bethesda Research Laboratories. Focus 8 , 9—12 Dietz, K.

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Molecular Cloning Central

Evaluation of seamless ligation cloning extract preparation methods from an Escherichia coli laboratory strain. A simple and ultra-low cost homemade seamless ligation cloning extract SLiCE as an alternative to a commercially available seamless DNA cloning kit. Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.

Expression of spinach ferredoxin-thioredoxin reductase using tandem T7 promoters and application of the purified protein for in vitro light-dependent thioredoxin-reduction system.

Key Steps of Molecular Cloning

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods. Sanger, F. DNA sequencing with chain-terminating inhibitors. Download references. The author read and approved the final manuscript. Correspondence to Ken Motohashi. Reprints and Permissions.

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Advanced search. Skip to main content. Subjects Genetic engineering PCR-based techniques. Abstract An efficient PCR cloning method is indispensable in modern molecular biology, as it can greatly improve the efficiency of DNA cloning processes.

Introduction Polymerase chain reaction PCR is an indispensable tool for amplification of genomic DNA and transcripts to analyze their functions 1. Figure 1. Full size image. Figure 2. Full size table. Table 2 Efficiencies with which blunt-end PCR products were cloned into the blunt-end vectors carrying the E. Figure 3. Figure 4. Figure 5. Outline of the PCR cloning processes of standard and rapid protocols. Figure 6. Table 5 Blunting and blunt-end cloning of the dA-tailed DNA fragments in the blunt-end positive-selection system using the ccdB gene.

Conclusion Three types of PCR cloning vectors were developed in this study. In silico cloning Serial Cloner 2. References 1. For in vivo cloning a fragment of DNA, containing a single gene or a number of genes, is inserted into a vector that can be amplified within another host cell.


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A vector is a section of DNA that can incorporate another DNA fragment without losing the capacity for self-replication, and a vector containing an additional DNA fragment is known as a hybrid vector. If the fragment of DNA includes one or more genes the process is referred to as gene cloning. The host cell copies the cloned DNA using its own replication mechanisms. A variety of cell types are used as hosts, including bacteria, yeast cells and mammalian cells. This is an in vitro method for making many copies of a specific section of DNA, without the need for vectors or host cells.

This enzyme is stable under high temperatures, and is obtained from the thermophilic bacterium Thermus aquaticus. The process involves the repetition of three steps:. Each cycle takes a few minutes, and repeated cycles can produce large amounts of a specific DNA sequence in a matter of hours rather than days. However, this cloning method does require knowledge of some details about the nucleotide sequence to be copied, and the technique is very sensitive to small amounts of contamination.

A gene library is a large collection of cloned DNA sequences from a single genome.

Library Hub Discover

These are used to investigate the structure of a given chromosome, or to clone specific genes. These types of libraries may be prepared from a subset of the entire genome for example, a single chromosome. The fragments are then linked to appropriate vectors and cloned in a suitable host cell population. The resulting cDNA represents the genes expressed in the cell population as a subset of the entire genome, and can be cloned using a vector and suitable host cell as seen in the diagram above. Restriction enzymes to cut the DNA and gel electrophoresis to separate the resulting fragments can be used to produce a physical map of DNA segments in a process known as restriction mapping.


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An example of what one of these may look like can be seen below.