DNA shuffling: Difference between revisions
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Several rounds of this PCR extension are allowed to occur, after some of the [[DNA]] molecules reach the size of the parental genes. These genes can then be amplified with another PCR, this time with the addition of [[Primer (molecular biology)|primers]] that are designed to complement the ends of the strands. The primers may have additional sequences added to their 5' ends, such as sequences for [[restriction enzyme]] recognition sites needed for ligation into a cloning vector. |
Several rounds of this PCR extension are allowed to occur, after some of the [[DNA]] molecules reach the size of the parental genes. These genes can then be amplified with another PCR, this time with the addition of [[Primer (molecular biology)|primers]] that are designed to complement the ends of the strands. The primers may have additional sequences added to their 5' ends, such as sequences for [[restriction enzyme]] recognition sites needed for ligation into a cloning vector. |
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It is possible to [[Genetic recombination|recombine]] portions of these genes to generate hybrids or [[Chimera (genetics)|chimeric forms]] with unique properties, hence the term DNA shuffling.<ref>{{cite journal|title=Anticipatory evolution and DNA shuffling|url= |
It is possible to [[Genetic recombination|recombine]] portions of these genes to generate hybrids or [[Chimera (genetics)|chimeric forms]] with unique properties, hence the term DNA shuffling.<ref>{{cite journal|title=Anticipatory evolution and DNA shuffling|url= |journal=Genome Biology |date=2002|volume=3|issue=8|pages=reviews1021.1|first1=Jamie M.|last1=Bacher|display-authors=etal|doi=10.1186/gb-2002-3-8-reviews1021|pmid=12186650|pmc=139397}}</ref> |
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==Shuffling methods== |
==Shuffling methods== |
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Revision as of 12:19, 25 April 2021
DNA shuffling is a way to rapidly propagate beneficial mutations in a directed evolution experiment.[1] It is used to rapidly increase DNA library size.[2]
Procedure
First, DNase is used to fragment a set of parent genes into pieces of 50-100 bp in length. This is then followed by a polymerase chain reaction (PCR) without primers- DNA fragments with sufficient overlapping homologous sequence will anneal to each other and are then extended by DNA polymerase.
Several rounds of this PCR extension are allowed to occur, after some of the DNA molecules reach the size of the parental genes. These genes can then be amplified with another PCR, this time with the addition of primers that are designed to complement the ends of the strands. The primers may have additional sequences added to their 5' ends, such as sequences for restriction enzyme recognition sites needed for ligation into a cloning vector.
It is possible to recombine portions of these genes to generate hybrids or chimeric forms with unique properties, hence the term DNA shuffling.[3]
Shuffling methods
Using restriction enzymes
- Restriction enzymes that cut in similar places are used to digest members of the gene family
- DNA fragments are joined together with DNA ligase
- Large numbers of hybrids that can be tested for unique properties are produced
Using DNAse 1
- Different members of the gene family are fragmented using DNase 1 followed by PCR
- During PCR different members of the family are cross-primed, i.e. homological DNA fragments will anneal to each other
- The generated hybrids are then used to generate a library of mutants, which are tested for unique properties
See also
References
- ^ Zhao, Huimin; Arnold, Frances H. (1997). "Optimization of DNA shuffling for high fidelity recombination" (PDF). Nucleic Acids Research. 25 (6): 1307–1308. doi:10.1093/nar/25.6.1307. PMC 146579. PMID 9092645.[permanent dead link]
- ^ Cohen, J. (13 July 2001). "How DNA Shuffling Works". Science. 293 (5528): 237. doi:10.1126/science.293.5528.237. PMID 11452110.
- ^ Bacher, Jamie M.; et al. (2002). "Anticipatory evolution and DNA shuffling". Genome Biology. 3 (8): reviews1021.1. doi:10.1186/gb-2002-3-8-reviews1021. PMC 139397. PMID 12186650.
{{cite journal}}: CS1 maint: unflagged free DOI (link)