DNA shuffling
[1][4] DNA shuffling utilizes random recombination as opposed to site-directed mutagenesis in order to generate proteins with unique attributes or combinations of desirable characteristics encoded in the parent genes such as thermostability and high activity.
[1][2] As a result of the random recombination, DNA shuffling is able to produce proteins with new qualities or multiple advantageous features derived from the parent genes.
[7] Since the introduction of the technique, DNA shuffling has been applied to protein and small molecule pharmaceuticals, bioremediation, vaccines, gene therapy, and evolved viruses.
[1][7] He started by fragmenting the β-lactamase gene that had been amplified with the polymerase chain reaction (PCR) by using DNase I, which randomly cleaves DNA.
[14] Stemmer reported that the use of DNA shuffling in combination with backcrossing resulted in the elimination of non-essential mutations and an increase in the production of the antibiotic cefotaxime.
[17][18][19] Additionally, DNA shuffling has been applied to protein and small molecule pharmaceuticals, bioremediation, gene therapy, vaccines, and evolved viruses.
[3][10][11][12][20] First, DNase I is used to fragment a set of parent genes into segments of double stranded DNA ranging from 10-50 bp to more than 1 kbp.
[1][14] It is possible to recombine portions of the parent genes to generate hybrids or chimeric forms with unique properties, hence the term DNA shuffling.
[1] The main advantages of using restriction enzymes include control over the number of recombination events and lack of PCR amplification requirement.
[3][27] For example, the homologous recombination method of DNA shuffling by molecular breeding has been utilized to enhance the detoxification of atrazine and arsenate.
[12][28] Specifically, a recombinant E. coli strain has been created with the use of DNA shuffling by molecular breeding for the bioremediation of trichloroethylene (TCE), a potential carcinogen, which is less susceptible to toxic epoxide intermediates.
[11] For example, DNA shuffling with molecular breeding was applied to six ecotropic murine leukemia virus (MLV) strains which resulted in the compilation of an extensive library of recombinant retrovirus and the identification of multiple clones with increased stability.
[11] Furthermore, the application of DNA shuffling by molecular breeding on multiple parent adeno-associated virus (AAV) vectors was employed to generate a library of ten million chimeras.
[29] The advantageous attributes obtained include increased resistance to human intravenous immunoglobulin (IVIG) and the production of cell tropism in the novel viruses.
[29] While DNA shuffling has become a useful technique for random recombination, other methods including RACHITT, RPR, and StEP have also been developed for this purpose.
[30] Some major benefits include the smaller requirement for parent genes due to the use of ss templates and increased sequence diversity by mispriming and misincorporation.
