DNA shuffling

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

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]

Contents

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]  

History

DNA shuffling by molecular breeding was first reported in 1994 by Willem P.C. Stemmer. [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] [15] He then completed a modified PCR reaction where primers were not employed which resulted in the annealing of homologous fragments or fragments with similar sequences. [14] Finally, these fragments were amplified by PCR. [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. [14] He also emphasized the potential for molecular evolution with DNA shuffling. [16] Specifically, he indicated the technique could be used to modify proteins. [16]    

DNA shuffling has since been applied to generate libraries of hybrid or chimeric genes and has inspired family shuffling which is defined as the use of related genes in DNA shuffling. [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]

Procedures

Molecular breeding

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. [4] [16] This is followed by a PCR without primers. [14] In the PCR, DNA fragments with sufficiently overlapping sequences will anneal to each other and then be extended by DNA polymerase. [1] [4] [5] [7] [14] [16] [21] The PCR extension will not occur unless there are DNA sequences of high similarity. [1] The important factors influencing the sequences synthesized in DNA shuffling are the DNA polymerase, salt concentrations, and annealing temperature. [10] For example, the use of Taq polymerase for amplification of a 1 kbp fragment in a PCR of 20 cycles results in 33% to 98% of the products containing one or more mutations. [21]

Multiple cycles of PCR extension can be used to amplify the fragments. [1] [4] [5] [7] [14] [16] [21] The addition of primers that are designed to be complementary to the ends of the extended fragments are added to further amplify the sequences with another PCR. [1] [14] [21] Primers may be chosen to have additional sequences added on to their 5’ ends, such as sequences for restriction enzyme recognition sites which are needed for ligation into a cloning vector. [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. [22] The disadvantage of molecular breeding is the requirement for the similarity between the sequences, which has inspired the development of other procedures for DNA shuffling. [1]

Restriction enzymes

Restriction enzymes are employed to fragment the parent genes. [1] [21] The fragments are then joined together through ligation which can be accomplished with DNA ligase. [1] For example, if two parent genes have three restriction sites fourteen different full-length gene hybrids can be created. [1] The number of unique full-length hybrids is determined by the fact that a gene with three restriction sites can be broken up into four fragments. [1] Thus, there are two options for each of the four positions minus the combinations that would recreate the two parent genes yielding 24 - 2 = 14 different full-length hybrid genes. [1]

The main difference between DNA shuffling with restriction enzymes and molecular breeding is molecular breeding relies on the homology of the sequences for the annealing of the strands and PCR for extension whereas by using restriction enzymes, fragment ends that can be ligated are created. [1] The main advantages of using restriction enzymes include control over the number of recombination events and lack of PCR amplification requirement. [1] [23] The main disadvantage is the requirement of common restriction enzyme sites. [1]

Nonhomologous random recombination

In order to generate segments ranging from 10-50 bp to more than 1 kb, DNase I is utilized. [4] [16] [24] The ends of the fragments are made blunt by adding T4 DNA polymerase. [1] [24] Blunting the fragments is important for combining the fragments as incompatible sticky-ends, or overhangs, prevent end joining. [1] [25] [26] Hairpins with a specific restriction site are then added to the mixture of fragments. [1] [24] Next, T4 DNA ligase is employed to ligate the fragments to form extended sequences. [1] [24] The ligation of the hairpins to the fragments limits the length of the extended sequences by preventing the addition of more fragments. [1] Finally, in order to remove the hairpin loops, a restriction enzyme is utilized. [1] [24]

Nonhomologous random recombination differs from molecular breeding as homology of the ligated sequences is not necessary which is an advantage. [1] However, because this process recombines the fragments randomly it is probable that a large fraction of the recombined DNA sequences will not have the desired characteristics which is a disadvantage. [1] Nonhomologous random recombination also differs from the use of restriction enzymes for DNA shuffling as common restriction enzyme sites on the parent genes are not required and the use of hairpins is necessary which demonstrates an advantage and disadvantage of nonhomologous random recombination over the use of restriction enzymes, respectively. [1]

Applications

Protein and small molecule pharmaceuticals

Since DNA shuffling enables the recombination of genes, protein activities can be enhanced. [3] [20] For example, DNA shuffling has been used to increase the potency of phage-displayed recombinant interferons on murine and human cells. [3] Additionally, the improvement of green fluorescent protein (GFP) was accomplished with DNA shuffling by molecular breeding as a 45-fold greater signal than the standard for whole cell fluorescence was obtained. [20] Furthermore, the synthesis of diverse genes can also result in the production of proteins with novel attributes. [1] Therefore, DNA shuffling has been used to develop proteins to detoxify chemicals. [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. [3] [27]  

Bioremediation

DNA shuffling has also been used to improve the degradation of biological pollutants. [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. [12] [21] [28]

Vaccines

The ability to select desirable recombinants with DNA shuffling has been used in combination with screening strategies to enhance vaccine candidates against infections with an emphasis on improving immunogenicity, vaccine production, stability, and cross-reactivity to multiple strains of pathogens. [3] [10] Some vaccine candidates for Plasmodium falciparum, dengue virus, encephalitic alphaviruses (including: VEEV, WEEV, and EEEV), human immunodeficiency virus-1 (HIV-1), and hepatitis B virus (HBV) have been investigated. [10]    

Gene therapy and evolved viruses

The requirements for human gene therapies include high purity, high-titer, and stability. [11] DNA shuffling allows for the fabrication of retroviral vectors with these attributes. [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]    

Comparison to other techniques

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. [4] [13] Below are some advantages and disadvantages of these other methods for recombination. [4] [7] [13]

RACHITT

In RACHITT, fragments of single stranded (ss) parent genes are annealed onto a ss template resulting in decreased mismatching which is an advantage. [13] Additionally, RACHIIT enables genes with low sequence similarity to be recombined. [7] However, a major disadvantage is the preparation of the ss fragments of the parent genes and ss template. [7] [13]

RPR

RPR makes use of random primers. [30] These random primers are annealed to template DNA and are then extended by the Klenow fragment. [30] Next, the templates are removed and the fragments are assembled by homology in a process similar to PCR. [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. [7] [30] One disadvantage of RPR is the preparation of the template. [30]  

StEP

In StEP, brief cycles of primer annealing to a template and extension by polymerase are employed to generate full-length sequences. [31] [32] The main advantages of StEP are the simplicity of the method and the lack of fragment purification. [7] [13] The disadvantages of StEP include that it is time consuming and requires sequence homology. [7] [13]

See also

Related Research Articles

<span class="mw-page-title-main">Complementary DNA</span> Single-stranded DNA synthesized from RNA

In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a specific protein in a cell that does not normally express that protein, or to sequence or quantify mRNA molecules using DNA based methods. cDNA that codes for a specific protein can be transferred to a recipient cell for expression, often bacterial or yeast expression systems. cDNA is also generated to analyze transcriptomic profiles in bulk tissue, single cells, or single nuclei in assays such as microarrays, qPCR, and RNA-seq.

<span class="mw-page-title-main">Polymerase chain reaction</span> Laboratory technique to multiply a DNA sample for study

The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation; Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.

<span class="mw-page-title-main">Primer (molecular biology)</span> Short strand of RNA or DNA that serves as a starting point for DNA synthesis

A primer is a short single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. DNA polymerase enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase. The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind. 

A restriction enzyme, restriction endonuclease, REase, ENase orrestrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

<span class="mw-page-title-main">Genetic recombination</span> Production of offspring with combinations of traits that differ from those found in either parent

Genetic recombination is the exchange of genetic material between different organisms which leads to production of offspring with combinations of traits that differ from those found in either parent. In eukaryotes, genetic recombination during meiosis can lead to a novel set of genetic information that can be further passed on from parents to offspring. Most recombination occurs naturally and can be classified into two types: (1) interchromosomal recombination, occurring through independent assortment of alleles whose loci are on different but homologous chromosomes ; & (2) intrachromosomal recombination, occurring through crossing over.

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<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

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<span class="mw-page-title-main">Molecular genetics</span> Scientific study of genes at the molecular level

Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. The field of study is based on the merging of several sub-fields in biology: classical Mendelian inheritance, cellular biology, molecular biology, biochemistry, and biotechnology. Researchers search for mutations in a gene or induce mutations in a gene to link a gene sequence to a specific phenotype. Molecular genetics is a powerful methodology for linking mutations to genetic conditions that may aid the search for treatments/cures for various genetics diseases.

<span class="mw-page-title-main">Library (biology)</span>

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<span class="mw-page-title-main">Recombinant DNA</span> DNA molecules formed by human agency at a molecular level generating novel DNA sequences

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

<span class="mw-page-title-main">Gene mapping</span> Process of locating specific genes

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<span class="mw-page-title-main">Directed evolution</span> Protein engineering method

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<span class="mw-page-title-main">Artificial gene synthesis</span> Group of methods in synthetic biology

Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing. This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.

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<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology

Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

The staggered extension process is a common technique used in biotechnology and molecular biology to create new, mutated genes with qualities of one or more initial genes.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Ligation (molecular biology)</span>

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References

  1. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 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. 1 2 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. 1 2 3 4 5 6 7 8 9 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. 1 2 3 4 5 6 7 8 9 10 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. 1 2 3 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. 1 2 3 4 5 6 7 8 9 10 11 12 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. 1 2 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. 1 2 3 4 5 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. 1 2 3 4 5 Powell SK, Kaloss MA, Pinkstaff A, McKee R, Burimski I, Pensiero M, et al. (December 2000). "Breeding of retroviruses by DNA shuffling for improved stability and processing yields". Nature Biotechnology. 18 (12): 1279–1282. doi:10.1038/82391. PMID   11101807. S2CID   1865270.
  12. 1 2 3 4 Rui L, Kwon YM, Reardon KF, Wood TK (May 2004). "Metabolic pathway engineering to enhance aerobic degradation of chlorinated ethenes and to reduce their toxicity by cloning a novel glutathione S-transferase, an evolved toluene o-monooxygenase, and gamma-glutamylcysteine synthetase". Environmental Microbiology. 6 (5): 491–500. doi:10.1111/j.1462-2920.2004.00586.x. PMID   15049922.
  13. 1 2 3 4 5 6 7 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.
  14. 1 2 3 4 5 6 7 8 9 Stemmer WP (August 1994). "Rapid evolution of a protein in vitro by DNA shuffling". Nature. 370 (6488): 389–391. Bibcode:1994Natur.370..389S. doi:10.1038/370389a0. PMID   8047147. S2CID   4363498.
  15. ↑ "DNase I (RNase-free) | NEB". www.neb.com. Retrieved 2021-10-30.
  16. 1 2 3 4 5 6 Stemmer WP (October 1994). "DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution". Proceedings of the National Academy of Sciences of the United States of America. 91 (22): 10747–10751. Bibcode:1994PNAS...9110747S. doi: 10.1073/pnas.91.22.10747 . PMC   45099 . PMID   7938023.
  17. ↑ Kikuchi M, Ohnishi K, Harayama S (2000-02-08). "An effective family shuffling method using single-stranded DNA". Gene. 243 (1): 133–137. doi:10.1016/S0378-1119(99)00547-8. ISSN   0378-1119. PMID   10675621.
  18. ↑ Crameri A, Raillard SA, Bermudez E, Stemmer WP (January 1998). "DNA shuffling of a family of genes from diverse species accelerates directed evolution". Nature. 391 (6664): 288–291. Bibcode:1998Natur.391..288C. doi:10.1038/34663. PMID   9440693. S2CID   4352696.
  19. ↑ Coco WM, Levinson WE, Crist MJ, Hektor HJ, Darzins A, Pienkos PT, et al. (April 2001). "DNA shuffling method for generating highly recombined genes and evolved enzymes". Nature Biotechnology. 19 (4): 354–359. doi:10.1038/86744. PMID   11283594. S2CID   35360374.
  20. 1 2 3 Crameri A, Whitehorn EA, Tate E, Stemmer WP (March 1996). "Improved green fluorescent protein by molecular evolution using DNA shuffling". Nature Biotechnology. 14 (3): 315–319. doi:10.1038/nbt0396-315. PMID   9630892. S2CID   22803570.
  21. 1 2 3 4 5 6 Zhao H, Arnold FH (March 1997). "Optimization of DNA shuffling for high fidelity recombination". Nucleic Acids Research. 25 (6): 1307–1308. doi:10.1093/nar/25.6.1307. PMC   146579 . PMID   9092645.
  22. ↑ Bacher JM, Reiss BD, Ellington AD (July 2002). "Anticipatory evolution and DNA shuffling". Genome Biology. 3 (8): REVIEWS1021. doi:10.1186/gb-2002-3-8-reviews1021. PMC   139397 . PMID   12186650.
  23. ↑ Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009-05-14). "Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes". PLOS ONE. 4 (5): e5553. Bibcode:2009PLoSO...4.5553E. doi: 10.1371/journal.pone.0005553 . PMC   2677662 . PMID   19436741.
  24. 1 2 3 4 5 Bittker JA, Le BV, Liu JM, Liu DR (May 2004). "Directed evolution of protein enzymes using nonhomologous random recombination". Proceedings of the National Academy of Sciences of the United States of America. 101 (18): 7011–7016. Bibcode:2004PNAS..101.7011B. doi: 10.1073/pnas.0402202101 . PMC   406457 . PMID   15118093.
  25. ↑ Zhu S, Peng A (June 2016). "Non-homologous end joining repair in Xenopus egg extract". Scientific Reports. 6 (1): 27797. Bibcode:2016NatSR...627797Z. doi:10.1038/srep27797. PMC   4914968 . PMID   27324260.
  26. ↑ Ogiwara H, Kohno T (2011-12-14). "Essential factors for incompatible DNA end joining at chromosomal DNA double strand breaks in vivo". PLOS ONE. 6 (12): e28756. Bibcode:2011PLoSO...628756O. doi: 10.1371/journal.pone.0028756 . PMC   3237495 . PMID   22194904.
  27. 1 2 Crameri A, Dawes G, Rodriguez E, Silver S, Stemmer WP (May 1997). "Molecular evolution of an arsenate detoxification pathway by DNA shuffling". Nature Biotechnology. 15 (5): 436–438. doi:10.1038/nbt0597-436. PMID   9131621. S2CID   25669058.
  28. 1 2 Canada KA, Iwashita S, Shim H, Wood TK (January 2002). "Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation". Journal of Bacteriology. 184 (2): 344–349. doi:10.1128/JB.184.2.344-349.2002. PMC   139589 . PMID   11751810.
  29. 1 2 Koerber JT, Jang JH, Schaffer DV (October 2008). "DNA shuffling of adeno-associated virus yields functionally diverse viral progeny". Molecular Therapy. 16 (10): 1703–1709. doi:10.1038/mt.2008.167. PMC   2683895 . PMID   18728640.
  30. 1 2 3 4 5 Esteban O, Woodyer RD, Zhao H (2003). "In vitro DNA recombination by random priming". In Arnold FH, Georgiou G (eds.). Directed Evolution Library Creation. Methods in Molecular Biology. Vol. 231. Totowa, NJ: Humana Press. pp. 99–104. doi:10.1385/1-59259-395-x:99. ISBN   978-1-59259-395-8. PMID   12824607.
  31. ↑ Zhao H, Giver L, Shao Z, Affholter JA, Arnold FH (March 1998). "Molecular evolution by staggered extension process (StEP) in vitro recombination". Nature Biotechnology. 16 (3): 258–261. doi:10.1038/nbt0398-258. PMID   9528005. S2CID   20490024.
  32. ↑ Aguinaldo AM, Arnold FH (2003). "Staggered extension process (StEP) in vitro recombination". In Arnold FH, Georgiou G (eds.). Directed Evolution Library Creation. Methods in Molecular Biology. Vol. 231. Totowa, NJ: Humana Press. pp. 105–110. doi:10.1385/1-59259-395-X:105. ISBN   978-1-59259-395-8. PMID   12824608.
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