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Genomics and Bionanotechnology
Published in Anil Kumar Anal, Bionanotechnology, 2018
The classical technique is convenient in the presence of well-defined restriction site in the sequence. On the other hand, this approach is not suitable in case of absence of restriction site and most of the eukaryotic genes are interrupted by intervening sequences (introns), which makes the gene of interest very large. As manipulation of the large genomic DNA is difficult due to size capacity of cloning vectors and multiple restriction endonucleases, alternative cloning approaches such as PCR-mediated TA cloning, ligation-independent cloning (LIC), recombinase-dependent cloning, and PCR-mediated cloning are being developed (Souii et al. 2013). In PCR-mediated TA cloning method, DNA fragment generated from PCR is directly stitched with vector without the use of any restriction enzymes. As DNA fragments with adenine (A) residue known as A-tailed DNA fragment and vectors with thymine (T) residue known as T-tailed are directly ligated, this approach is known as TA cloning.LIC is done by adding short sequence of DNA to the insert DNA, which is homologous to the vector. Complementary cohesive ends between the insert and vector are formed by the enzymes with 3′–5′ exonuclease activity and the resulting two molecules are annealed together such that plasmid will have four single-stranded DNA nicks.The seamless cloning method allows sequence-independent and scar-free, that is, free of restriction enzyme sites and unwanted sequence, insertion of DNA insert into vector. This method includes addition of homologous region at each end of insert to be cloned and with action of enzyme exonuclease, DNA polymerase and ligase-recombinant DNA are formed.Recombinatorial cloning technique utilizes site-specific DNA recombinase enzymes that can swap pieces of DNA between two molecules with appropriate recombination site. Initially, the recombination sites are added on either side of insert by PCR followed by recombination with vector to make an entry clone, which is further recombined with destination vector to develop final construct. Gateway cloning system is one of the widely used cloning techniques based on this approach (Bertero et al. 2017).
Predicting algorithm of attC site based on combination optimization strategy
Published in Connection Science, 2022
Zhendong Liu, Xi Chen, Dongyan Li, Xinrong Lv, Mengying Qin, Ke Bai, Zhiqiang He, Yurong Yang, Xiaofeng Li, Qionghai Dai
Gene recombination is a way that organisms use recombinase to recombine different genes to produce new genotype individuals. It is widely present in prokaryotes and has important meanings such as maintaining biological genetic diversity and promoting biological evolution (Epum & Haber, 2022). Common recombination includes: homologous recombination, translocation recombination and site-specific recombination. Currently, with the development of site-specific recombination systems, site-specific recombination technology has been extensively used in various biological genetic engineering operations, especially in higher eukaryotes (Bessen et al., 2019; Häcker et al., 2017). Site-specific recombination refers to the integration, excision and transformation of DNA fragments between specific sites, which is catalysed by integron integrase. This type of recombination is associated with specific DNA sequences in bacteriophages and bacteria, and the reaction always involves two DNA-specific sites. However, these two specific sites usually have very similar or even exactly the same DNA sequences. Such sequence-level constraints restrict efficient recombination between the two sites (Tian & Zhou, 2021). Therefore, in order to solve the problem of sequence constraints, it is necessary to study the structure of specific recombination sites in the recombination system.