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A Guide to Gibson Assembly from the University of Cambridge, UK; Gibson Assembly Primer Design Tool; Gibson Assembly Site Directed Mutagenesis Primer Design Tool; Chemical Transformation of Gibson Assembly Constructs; Perkel, Jeffrey M. (January 2014). "Seamlessly rewriting the lab cloning manual". Tech News. BioTechniques. 56 (1): 12– 14.
The Gibson assembly method is a relatively straightforward DNA assembly method, requiring only a few additional reagents: the 5' T5 exonuclease, Phusion DNA polymerase, and Taq DNA ligase. The DNA fragments to be assembled are synthesised to have overlapping 5' and 3' ends in the order that they are to be assembled in.
Gibson assembly is a quick cloning method that uses three primary enzymes; 5' exonuclease, polymerase and ligase. [15] The exonuclease digests the 5' end of DNA fragments leaving a 3' overhang. [15] If there is significant homology (20-40 bp) on each end of the DNA insert, it can anneal with a complementary backbone. [15]
The overall success of OE-PCR based DNA assemblies relies on several factors, being the most relevant ones the instrinsic features of the DNA sequence to assemble, the sequence and length of the overlapping overhangs, the design of outer primers for the final amplification and the conditions of the PCR reaction.
The BioBrick assembly standard is a more reliable approach for combining parts to form larger composites. The assembly standard enables two groups of synthetic biologists in different parts of the world to re-use a BioBrick part without going through the whole cycle of design and manipulation. [2]
Golden Gate assembly involves digesting DNA sequences containing a type IIS restriction enzyme cut site and ligating them together. Golden Gate Cloning or Golden Gate assembly [1] is a molecular cloning method that allows a researcher to simultaneously and directionally assemble multiple DNA fragments into a single piece using Type IIS restriction enzymes and T4 DNA ligase. [2]
However, the genomes capable of being synthesized using this method alone is limited because as DNA cassettes increase in length, they require propagation in vitro in order to continue hybridizing; accordingly, Gibson assembly is often used in conjunction with Transformation-Associated Recombination (see below) to synthesize genomes several ...
Saturation mutagenesis is commonly achieved by site-directed mutagenesis PCR with a randomised codon in the primers (e.g. SeSaM) [2] or by artificial gene synthesis, with a mixture of synthesis nucleotides used at the codons to be randomised. [3] Different degenerate codons can be used to encode sets of amino acids. [1]