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DNA shuffling

Point mutations result in single nucleotide changes whereas insertions and deletions result in the addition or removal of nucleotides, respectively.[1][2] DNA shuffling enables the recombination of parent genes which dramatically increases the rate of directed evolution.[3] DNA shuffling is useful for generating proteins with novel properties or combinations of desired properties.[1]

DNA shuffling, also known as molecular breeding, is an in vitro random recombination method to generate mutant genes for directed evolution and to enable a rapid increase in DNA library size.[1][3][4][5][6][7][8][9] Three procedures for accomplishing DNA shuffling are molecular breeding which relies on homologous recombination or the similarity of the DNA sequences, restriction enzymes which rely on common restriction sites, and nonhomologous random recombination which requires the use of hairpins.[1] In all of these techniques, the parent genes are fragmented and then recombined.[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][8] The potential for DNA shuffling to produce novel proteins is exemplified by the figure shown on the right which demonstrates the difference between point mutations, insertions and deletions, and DNA shuffling.[1] Specifically, this figure shows the use of DNA shuffling on two parent genes which enables the generation of recombinant proteins that have a random combination of sequences from each parent gene.[1] This is distinct from point mutations in which one nucleotide has been changed, inserted, or deleted and insertions or deletions where a sequence of nucleotides has been added or removed, respectively.[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.[1][4]

In 1994, Willem P.C. Stemmer published the first paper on DNA shuffling.[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.[3][10][11][12] Other techniques which yield similar results to DNA shuffling include random chimeragenesis on transient templates (RACHITT), random printing in vitro recombination (RPR), and the staggered extension process (StEP).[4][7][13]  

  1. ^ a b c d e f g h i j Glick BR (2017). Molecular biotechnology : principles and applications of recombinant DNA. Cheryl L. Patten (Fifth ed.). Washington, DC. ISBN 978-1-55581-936-1. OCLC 975991667.{{cite book}}: CS1 maint: location missing publisher (link)
  2. ^ a b Levi T, Sloutskin A, Kalifa R, Juven-Gershon T, Gerlitz O (2020-07-14). "Efficient In Vivo Introduction of Point Mutations Using ssODN and a Co-CRISPR Approach". Biological Procedures Online. 22 (1): 14. doi:10.1186/s12575-020-00123-7. PMC 7362497. PMID 32684853.
  3. ^ a b c Patten PA, Howard RJ, Stemmer WP (December 1997). "Applications of DNA shuffling to pharmaceuticals and vaccines". Current Opinion in Biotechnology. 8 (6): 724–733. doi:10.1016/S0958-1669(97)80127-9. PMID 9425664.
  4. ^ a b c d Cirino PC, Qian S (2013-01-01), Zhao H (ed.), "Chapter 2 - Protein Engineering as an Enabling Tool for Synthetic Biology", Synthetic Biology, Boston: Academic Press, pp. 23–42, doi:10.1016/b978-0-12-394430-6.00002-9, ISBN 978-0-12-394430-6
  5. ^ Clark DP, Pazdernik NJ (2016), "Protein Engineering", Biotechnology, Elsevier, pp. 365–392, doi:10.1016/b978-0-12-385015-7.00011-9, ISBN 978-0-12-385015-7
  6. ^ Kamada H (2013-01-01), Park K, Tsunoda SI (eds.), "5 - Generating functional mutant proteins to create highly bioactive anticancer biopharmaceuticals", Biomaterials for Cancer Therapeutics, Woodhead Publishing, pp. 95–112, doi:10.1533/9780857096760.2.95, ISBN 978-0-85709-664-7
  7. ^ a b c Bioprocessing for value-added products from renewable resources : new technologies and applications. Shang-Tian Yang (1st ed.). Amsterdam: Elsevier. 2007. ISBN 978-0-444-52114-9. OCLC 162587118.{{cite book}}: CS1 maint: others (link)
  8. ^ a b Arkin M (2001-01-01), "In vitro Mutagenesis", in Brenner S, Miller JH (eds.), Encyclopedia of Genetics, New York: Academic Press, pp. 1010–1014, doi:10.1006/rwgn.2001.0714, ISBN 978-0-12-227080-2
  9. ^ Marshall SH (November 2002). "DNA shuffling: induced molecular breeding to produce new generation long-lasting vaccines". Biotechnology Advances. 20 (3–4): 229–238. doi:10.1016/s0734-9750(02)00015-0. PMID 14550030.
  10. ^ Locher CP, Paidhungat M, Whalen RG, Punnonen J (April 2005). "DNA shuffling and screening strategies for improving vaccine efficacy". DNA and Cell Biology. 24 (4): 256–263. doi:10.1089/dna.2005.24.256. PMID 15812242.
  11. ^ Cite error: The named reference :14 was invoked but never defined (see the help page).
  12. ^ Cite error: The named reference :10 was invoked but never defined (see the help page).
  13. ^ Kurtzman AL, Govindarajan S, Vahle K, Jones JT, Heinrichs V, Patten PA (August 2001). "Advances in directed protein evolution by recursive genetic recombination: applications to therapeutic proteins". Current Opinion in Biotechnology. 12 (4): 361–370. doi:10.1016/S0958-1669(00)00228-7. PMID 11551464.
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