Dna Recombination In Genetic Engineering

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Abstract DNA recombination is basically generation of the new DNA sequence in genome by exchange of DNA strands. Recombination generally but not necessarily occurs between similar DNA sequences and provides genetic variation, genome integrity [1]. There are four main ways being identified to produce recombinant DNA; homologous recombination in which physical exchange of DNA sequences occur between the homologous chromosomes, illegitimate recombination occurs between DNA sequences sharing low sequence similarity, site specific recombination require recombination enzymes that specifically bound short nucleotide sequence where cleave and rejoining occurs. The last class is transposition in which mobile DNA element move from one location to target …show more content…
Transposition shows little sequence selectivity unlike the homologous recombination for insertion but require specific enzymes to move the different locations within genome. Also, transposition (class I) is widely resulting in duplication of original DNA sequences. There are considerable diversity among transposition elements and mainly classified into two groups according to their movement mechanisms. Class I is retro-transposons which use a RNA intermediate in transposition mechanism and Class II is DNA transposons remaining as DNA throughout transposition [4]. Retro transposons first transcribe themselves in RNA and then RNA is copied to DNA again by reverse transcriptase which is generally encoded by mobile elements. Copied DNA is inserted into target site. The main class of retro transposons are virus like and poly A retro transposons. On the other hand, the distinctive property of Class II mechanisms which generally use cut and paste mechanism does not involve RNA intermediated in their transposition. The enzyme called transposase cut the target sequence and generates sticky ends. The transposon which is cut out is then ligated to acceptor sequence. There are also DNA transposons such as Tn3 and IS1 which copy themselves into target site rather than cut/paste and cause replication of donor …show more content…
cerevisiae and drosophila (Rothstein 1991; S. Rong 2002). Especially in yeast cells, HR is commonly used method for gene targeting due to high efficiency. Yeast cells can grow as haploid/diploid forms; it provides specific advantage to study lethality-gene relationship and yeast mating type switch stimulates the efficient homologous recombination machinery. Target gene can be disrupted by using plasmid containing nonfunctional copy of gene, selectable marker flanked by short ~40 nucleotides homologous to target gene [5]. Basically, nonfunctional copy of gene is cloned into plasmid vector containing selectable marker. Then vector transformed into yeast cells integrates target site in the genome by HR. The major advantage is that this methodology is systematically efficient enough to mutagenize annotated ~6200 yeast ORFs due to known sequence information. Nevertheless, frequency of HR could be increased by introducing double strand break (DSB) within the plasmid sequence having homology with target gene. DSB typically repaired by non-homologous end joining or HR which cause gene conversion. Orr-Weaver et al. signified that the position of double strand break is important factor and affect frequency of genetic exchange [6]. Afterwards, it was shown that recombination frequency is increasing when circular vector is linearized (Orr-Weaver,

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