Editing Restriction enzyme (section)

==Applications==
{{Main|Restriction digest}}
Isolated restriction enzymes are used to manipulate DNA for different scientific applications.

They are used to assist insertion of genes into [[plasmid vector]]s during [[gene cloning]] and [[protein production]] experiments. For optimal use, plasmids that are commonly used for gene cloning are modified to include a short polylinker sequence (called the [[multiple cloning site]], or MCS) rich in restriction [[recognition sequence]]s. This allows flexibility when inserting gene fragments into the plasmid vector; restriction sites contained naturally within genes influence the choice of endonuclease for digesting the DNA, since it is necessary to avoid restriction of wanted DNA while intentionally cutting the ends of the DNA. To clone a gene fragment into a vector, both plasmid DNA and gene insert are typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme known as a [[DNA ligase]].<ref name="urlCloning using restriction enzymes">{{cite web | url = http://www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/restriction_enzymes/ | title = Cloning using restriction enzymes | author = Geerlof A | publisher = European Molecular Biology Laboratory - Hamburg | access-date = 2008-06-07}}</ref><ref name="isbn0-87969-576-5">{{cite book | vauthors = Russell DW, Sambrook J | title = Molecular cloning: a laboratory manual | publisher = Cold Spring Harbor Laboratory | location = Cold Spring Harbor, N.Y | year = 2001 | isbn = 0-87969-576-5 | url-access = registration | url = https://archive.org/details/molecularcloning0000samb_p7p5 }}</ref>

Restriction enzymes can also be used to distinguish gene [[allele]]s by specifically recognizing single base changes in DNA known as [[single-nucleotide polymorphism]]s (SNPs).<ref name="pmid18330346">{{cite journal | vauthors = Wolff JN, Gemmell NJ | title = Combining allele-specific fluorescent probes and restriction assay in real-time PCR to achieve SNP scoring beyond allele ratios of 1:1000 | journal = BioTechniques | volume = 44 | issue = 2 | pages = 193–4, 196, 199 | date = February 2008 | pmid = 18330346 | doi = 10.2144/000112719 | doi-access = free }}</ref><ref name="pmid15980518">{{cite journal | vauthors = Zhang R, Zhu Z, Zhu H, Nguyen T, Yao F, Xia K, Liang D, Liu C | display-authors = 6 | title = SNP Cutter: a comprehensive tool for SNP PCR-RFLP assay design | journal = Nucleic Acids Research | volume = 33 | issue = Web Server issue | pages = W489-92 | date = July 2005 | pmid = 15980518 | pmc = 1160119 | doi = 10.1093/nar/gki358 }}</ref> This is however only possible if a SNP alters the restriction site present in the allele. In this method, the restriction enzyme can be used to [[genotype]] a DNA sample without the need for expensive [[DNA sequencing|gene sequencing]]. The sample is first digested with the restriction enzyme to generate DNA fragments, and then the different sized fragments separated by [[gel electrophoresis]]. In general, alleles with correct restriction sites will generate two visible bands of DNA on the gel, and those with altered restriction sites will not be cut and will generate only a single band. A [[restriction map|DNA map]] by restriction digest can also be generated that can give the relative positions of the genes.<ref>{{cite web |url=http://www.nature.com/scitable/definition/mapping-282 |title=Mapping |work=Nature }}</ref>  The different lengths of DNA generated by restriction digest also produce a specific pattern of bands after gel electrophoresis, and can be used for [[DNA fingerprinting]].

In a similar manner, restriction enzymes are used to digest [[genomic]] DNA for gene analysis by [[Southern blot]]. This technique allows researchers to identify how many copies (or [[paralogue]]s) of a gene are present in the genome of one individual, or how many gene [[mutation]]s ([[Polymorphism (biology)|polymorphisms]]) have occurred within a population. The latter example is called [[Restriction Fragment Length Polymorphism|restriction fragment length polymorphism]] (RFLP).<ref name="isbn0-7167-4684-0">{{cite book |vauthors=Stryer L, Berg JM, Tymoczko JL | title = Biochemistry | edition = Fifth | publisher = W.H. Freeman | location = San Francisco | year = 2002 | page = 122 | isbn = 0-7167-4684-0}}</ref>

Artificial restriction enzymes created by linking the FokI DNA cleavage domain with an array of DNA binding proteins or zinc finger arrays, denoted zinc finger nucleases (ZFN), are a powerful tool for host genome editing due to their enhanced sequence specificity. ZFN work in pairs, their dimerization being mediated in-situ through the FokI domain. Each zinc finger array (ZFA) is capable of recognizing 9–12 base pairs, making for 18–24 for the pair. A 5–7 bp spacer between the cleavage sites further enhances the specificity of ZFN, making them a safe and more precise tool that can be applied in humans. A recent Phase I clinical trial of ZFN for the targeted abolition of the CCR5 co-receptor for HIV-1 has been undertaken.<ref>{{cite journal | vauthors = Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G, Spratt SK, Surosky RT, Giedlin MA, Nichol G, Holmes MC, Gregory PD, Ando DG, Kalos M, Collman RG, Binder-Scholl G, Plesa G, Hwang WT, Levine BL, June CH | display-authors = 6 | title = Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV | journal = The New England Journal of Medicine | volume = 370 | issue = 10 | pages = 901–10 | date = March 2014 | pmid = 24597865 | pmc = 4084652 | doi = 10.1056/NEJMoa1300662 }}</ref>

Others have proposed using the bacteria R-M system as a model for devising human anti-viral gene or genomic vaccines and therapies since the RM system serves an innate defense-role in bacteria by restricting tropism by bacteriophages.<ref>{{cite journal|author=Wayengera M |title= HIV and Gene Therapy: The proposed [R-M enzymatic] model for a gene therapy against HIV. |journal=Makerere Med J. |year=2003 |volume=38 |pages=28–30}}</ref> There is research on REases and ZFN that can cleave the DNA of various human viruses, including [[Herpes simplex virus|HSV-2]], high-risk [[HPV]]s and [[HIV-1]], with the ultimate goal of inducing target mutagenesis and aberrations of human-infecting viruses.<ref>{{cite journal |vauthors=Wayengera M, Kajumbula H, Byarugaba W |title= Frequency and site mapping of HIV-1/SIVcpz, HIV-2/SIVsmm and Other SIV gene sequence cleavage by various bacteria restriction enzymes: Precursors for a novel HIV inhibitory product |journal= Afr J Biotechnol |year=2007 |volume= 6 |issue=10 |pages=1225–1232 }}</ref><ref>{{cite journal | vauthors = Schiffer JT, Aubert M, Weber ND, Mintzer E, Stone D, Jerome KR | title = Targeted DNA mutagenesis for the cure of chronic viral infections | journal = Journal of Virology | volume = 86 | issue = 17 | pages = 8920–36 | date = September 2012 | pmid = 22718830 | pmc = 3416169 | doi = 10.1128/JVI.00052-12 }}</ref><ref>{{cite journal | vauthors = Manjunath N, Yi G, Dang Y, Shankar P | title = Newer gene editing technologies toward HIV gene therapy | journal = Viruses | volume = 5 | issue = 11 | pages = 2748–66 | date = November 2013 | pmid = 24284874 | pmc = 3856413 | doi = 10.3390/v5112748 | doi-access = free }}</ref> The human genome already contains remnants of retroviral genomes that have been inactivated and harnessed for self-gain. Indeed, the mechanisms for silencing active L1 genomic retroelements by the three prime repair exonuclease 1 (TREX1) and excision repair cross complementing 1(ERCC) appear to mimic the action of RM-systems in bacteria, and the non-homologous end-joining (NHEJ) that follows the use of ZFN without a repair template.<ref>{{cite journal | vauthors = Stetson DB, Ko JS, Heidmann T, Medzhitov R | title = Trex1 prevents cell-intrinsic initiation of autoimmunity | journal = Cell | volume = 134 | issue = 4 | pages = 587–98 | date = August 2008 | pmid = 18724932 | pmc = 2626626 | doi = 10.1016/j.cell.2008.06.032 }}</ref><ref>{{cite journal | vauthors = Gasior SL, Roy-Engel AM, Deininger PL | title = ERCC1/XPF limits L1 retrotransposition | journal = DNA Repair | volume = 7 | issue = 6 | pages = 983–9 | date = June 2008 | pmid = 18396111 | pmc = 2483505 | doi = 10.1016/j.dnarep.2008.02.006 }}</ref>