Image EcoRI restriction enzyme recognition site.svg thumb 175px EcoRI recognition site with cutting pattern indicated by a green line File Ecor1 2ckq.png thumb EcoRI crystal structure. Dimer bound to DNA Protein PDB PDBe 1ckq Infobox protein family Symbol EcoRI Name EcoRI image PDB 1qc9 EBI.jpg width caption The crystallographic structure of restriction endonuclease EcoRI at 3.3 a in the absence of DNA Pfam PF02963 Pfam clan InterPro IPR004221 SMART PROSITE MEROPS SCOP 1na6 TCDB OPM family OPM protein CAZy CDD Eco RI pronounced, eco R one or, rarely and wrongly, eco Rye is an endonuclease enzyme isolated from strains of E. coli , and is part of the restriction modification system . In molecular biology it is used as a restriction enzyme . It creates DNA end sticky ends with 5 end overhangs. The nucleic acid sequence where the enzyme cuts is GAATTC, which, as the complementary sequence is CTTAAG, has rotational symmetry . Structure Primary structure EcoRI contains the PD..D EXK motif within its active site like many Restriction enzyme restriction endonucleases . It is typically used in the isolation and restriction of bacterial plasmid DNA. In EcoRI this motif consists of residues P90 ... which sticks out from the globular domain and wraps around the DNA when bound 3 . EcoRI has been ... to allow the enzyme s two active sites to communicate 4 . Uses Restriction enzymes such as EcoRI ... , DNA screening and deleting sections of DNA in vitro . Restriction enzymes like EcoRI that generate ... ends make the DNA ligase ligation reaction more efficient. EcoRI can exhibit non site ... that can induce star activity when using EcoRI include low salt concentration, high glycerol ... title Mechanisms of coupling between DNA recognition and catalysis in EcoRI endonucleases journal ... Category Restriction enzymes hydrolase stub de EcoRI es EcoRI fr EcoRI he EcoRI ja EcoRI pl EcoRI sv EcoRI zh EcoRI ... more details
Orphan date April 2012 pSC101 is a DNA plasmid that is used as a cloning Vector molecular biology vector in genetic cloning experiments. pSC101 was the first cloning vector, used in 1973 by Herbert Boyer and Stanley Norman Cohen . They demonstrated that a gene from a frog could be transferred into bacterial cells and then expressed by the bacterial cells. History In the early 1970s, ref name Introduction to Biotechnology cite book title Introduction to Biotechnology author Thieman, W.J. and Palladino, M.A. year 2004 publisher Pearson Education, Benjamin Cummings page 55 ref Herbert Boyer and Stanley Norman Cohen produced pSC101, the first plasmid vector for cloning purposes. Soon after successfully cloning two pSC101 plasmids together to create one large plasmid, they published the results describing the experiment, in 1973. ref name Introduction to Biotechnology The cloning of genes into plasmids occurred soon after. In 1980, ref name Introduction to Biotechnology pSC101 became the first patented commercial DNA cloning vector when patents were awarded to Boyer and Cohen. The SC stands for Stanley Cohen. Although the original pSC101 only contained tetracycline resistance and a restriction site for EcoRI, the commercially available pSC101 gained restriction sites for several enzymes, including HindIII, in addition to the EcoRI site. References Reflist Category Molecular genetics genetics stub ... more details
Restriction sites , or restriction recognition sites , are locations on a DNA molecule containing specific 4 8 base pairs in length ref iGenetics A Mendelian Approach, Peter Russell, 2006 ref sequences of nucleotide s, which are recognized by restriction enzyme s. These are generally palindromic sequence s ref Cite book title Principles of Biochemistry edition 5th first1 Albert L. first2 David L. first3 Michael M. last1 Lehninger last2 Nelson last3 Cox publisher W.H. Freeman and Company location New York, NY year 2008 isbn 978 0 7167 7108 1 page 305 ref because restriction enzymes usually bind as homodimer s , and a particular restriction enzyme may cut the sequence between two nucleotides within its recognition site, or somewhere nearby. For example, the common restriction enzyme EcoRI recognizes the palindromic sequence GAATTC and cuts between the G and the A on both the top and bottom strands, leaving an overhang an end portion of a DNA strand with no attached complement known as a sticky end ref Cite book title Principles of Biochemistry edition 5th first1 Albert L. first2 David L. first3 Michael M. last1 Lehninger last2 Nelson last3 Cox publisher W.H. Freeman and Company location New York, NY year 2008 isbn 978 0 7167 7108 1 page 306 ref on each end, of AATT. This overhang can then be used to ligate in see DNA ligase a piece of DNA with a complementary overhang another EcoRI cut piece, for example . Some restriction enzymes cut DNA at a restriction site in a manner which leaves no overhang, a blunt end . ref Cite book title Principles of Biochemistry edition 5th first1 Albert L. first2 David L. first3 Michael M. last1 Lehninger last2 Nelson last3 Cox publisher W.H. Freeman and Company location New York, NY year 2008 isbn 978 0 7167 7108 1 page 306 ref References Reflist DEFAULTSORT Restriction Site Category Molecular biology molecular cell biology stub fr Sites de restriction it Sito di restrizione ru ... more details
of an insert cloned with EcoRI will be found. Digests 1 EcoRI 2 HindIII 3 EcoRI HindIII optional and not discussed ... Multiple Cloning Site of Vector 5 HindIII EcoRI 3 Discussion The EcoRI digest excises the insert ... more details
EcoRI Eco RI comes from Escherichia coli RY13 bacteria, while HindII comes from Haemophilus influenzae ... isolated from single strains of bacteria EcoRI Eco RI , EcoRII Eco RII . Nucleases are further described .... For example, the nuclease EcoRI has the following recognition sequence class wikitable Enzyme Source Recognition Sequence Cut EcoRI Eco RI Escherichia coli center 5 GAATTC br 3 CTTAAG center center ... AE AEC CC action.html Restriction Enzyme Action of EcoRI http www.accessexcellence.org AE ... more details
for the restriction enzyme EcoRI and a gene for tetracycline resistance. The restriction enzyme EcoRI ..., which had also been cleaved with EcoRI. The sticky end s of the DNA segments aligned ... more details
A multiple cloning site MCS , also called a polylinker , is a short segment of DNA which contains many up to 20 restriction sites a standard feature of engineered plasmids . ref cite book author Clark DP title Molecular Biology pages 611 publisher Academic Press year 2005 isbn 0121755517 url http books.google.com books?id 9Hw mstILcIC&pg PA611&lpg PA611&source bll&ots TxuRiQgOBQ&sig DBE9YCJJCO5Oc0PwLxAnGLheffM&hl en&ei qr2vSqjHKo6iMfO94PIN&sa X&oi book result&ct result&resnum 13 v onepage ref Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during procedures involving molecular cloning or subcloning. Extremely useful in biotechnology , bioengineering , and molecular genetics, MCSs let a biotechnologist insert a piece of DNA or several pieces of DNA into the region of the MCS. This can be used to create transgenic organisms, also known as genetically modified organisms GMOs . The pUC18 and pUC19 polylinker One bacterial plasmid used in genetic engineering as a plasmid cloning vector is pUC18 . Its polylinker region is composed of several restriction enzyme recognition sites, that have been engineered into a single cluster the polylinker . It has restriction sites for various restriction enzymes, including EcoRI , BamHI and PstI . Another vector used in genetic engineering is pUC19 , which is similar to pUC18, but its polylinker region is reversed. References reflist External links Category Genetics genetics stub de Polylinker pl Polilinker sv Multiple cloning site ... more details
Star activity is a relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimum for the enzyme. The result is typically cleavage at non canon basic principle canon ical recognition site, or sometimes complete loss of specificity. Differences which can lead to star activity include low ionic strength , high pH , and high 5 v v glycerol concentrations ref Robinson CR and Sliger SG 1993 Molecular Recognition Mediated by Bound Water A Mechanism for Star Activity of the Restriction Endonuclease EcoRI. Journal of Molecular Biology. 234 302 306. ref . The latter condition is of particular practical interest, since commercial restriction enzymes are usually supplied in a Buffer solution buffer containing a substantial amount of glycerol 50 v v is typical , meaning insufficient dilution of the enzyme solution can cause star activity this problem most often arises during double or multiple digests. Star activity can happen because of presence of Mg sup 2 sup , as is seen in HindIII , for example. External links http www.neb.com nebecomm tech reference restriction enzymes star activity.asp Star Activity New England Biolabs http www.fermentas.com techinfo re restrstaract.htm Star Activity Relaxation of Specificity Fermentas http bio.takara.co.jp BIO EN catalog d.asp?C ID C0008 Star activity of restriction enzymes a detailed list from TaKaRa http www.pubmedcentral.nih.gov articlerender.fcgi?artid 2396408 The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases References Reflist enzyme stub Category Restriction enzymes Category DNA de Star Aktivit t pl Aktywno star zh ... more details
Infobox protein family Symbol BamHI Name BamHI image PDB 1esg EBI.jpg width caption restriction endonuclease bamhi bound to a non specific dna. Pfam PF02923 Pfam clan CL0236 InterPro IPR004194 SMART PROSITE MEROPS SCOP 1bhm TCDB OPM family OPM protein CAZy CDD BamHI is a restriction enzyme , derived from Bacillus amyloliquefaciens . It has the recognition site G GATCC , and leaves a sticky end . ref Molecular cell biology. Lodish, Harvey F. 5. ed. New York W. H. Freeman and Co., 2003, 973 s. b ill. ISBN 0 7167 4366 3 ref ref name pmid8145855 cite journal author Newman M, Strzelecka T, Dorner LF, Schildkraut I, Aggarwal AK title Structure of restriction endonuclease BamHI and its relationship to EcoRI journal Nature volume 368 issue 6472 pages 660 4 year 1994 month April pmid 8145855 doi 10.1038 368660a0 url ref ref name pmid10882125 cite journal author Viadiu H, Aggarwal AK title Structure of BamHI bound to nonspecific DNA a model for DNA sliding journal Mol. Cell volume 5 issue 5 pages 889 95 year 2000 month May pmid 10882125 doi 10.1016 S1097 2765 00 80329 9 url ref One of the earlier enzymes to be used, it is popular for historical reasons, but also because digestion leaves a GATC overhang compatible with many other enzymes. Persistent issues with this specific enzyme spontaneously failing to generate cloneable fragments necessitate aliquoting prior to use. Engineered variants of the enzyme have been created in order to avoid star activity . Recognition site div style font size 14pt G span style border left 1px solid green border bottom 1px solid green G span span style border bottom 1px solid green A T C span C C C T A span style border right 1px solid green G span G div As at the end of 2010, there were http www.ebi.ac.uk pdbe srv view search index ?Uniprot accession P23940 5 crystal structures of BamH1 in the Protein Data Bank References references External links MeshName Deoxyribonuclease BamHI InterPro content IPR004194 DEFAULTSORT Bamhi Category Molecular ... more details
Refimprove date December 2009 Endonucleases are enzyme s that cleave the phosphodiester bond within a polynucleotide chain, in contrast to exonuclease s, which cleave phosphodiester bond s at the end of a polynucleotide chain. Typically, a restriction site will be a palindromic sequence four to six nucleotides long. Most restriction endonucleases cleave the DNA strand unevenly, leaving complementary single stranded ends. These ends can reconnect through hybridization and are termed sticky ends. Once paired, the phosphodiester bonds of the fragments can be joined by DNA ligase. There are hundreds of restriction endonucleases known, each attacking a different restriction site. A given sample of DNA is likely to contain a recognition sequence for any restriction endonuclease. The DNA fragments cleaved by the same endonuclease can be joined together regardless of the origin of the DNA. Such DNA is called recombinant DNA it has been artificially recombined Citation needed date February 2012 . Restriction endonucleases restriction enzyme s and are divided into three categories, Type I, Type II, and Type III, according to their mechanism of action. These enzymes are often used in genetic engineering to make recombinant DNA for introduction into bacterial, plant, or animal cells, as well as in synthetic biology . ref Emergent computation Emphasizing Bioinformatics , by Matthew Simon, Springer, Appendix, pp. 375 390 ref The commonly used notation for restriction endonucleases is of the form vwxyz , where vwx names the life form bacteria where this restriction endonuclease may be found, y names the strain and is optional , and z in Roman numerals indicates different restriction endonucleases in the same life form bacteria . Thus for example, EcoRI means that the restriction endonuclease is found in Escherichia coli Eco strain RY13 R , restriction endonuclease number I . Another example HaeII and HaeIII refer to bacterium Haemophilus aegyptius , number II and number III, respe ... more details
that the bacteriophage P1 had a unique site specific recombination system. EcoRI fragments ... lambda vectors . A 6.5x10 sup 3 sup base EcoRI fragment Fragment 7 was found to permit efficient ... more details
importance than mirror like palindromes. EcoRI digestion produces sticky ends, Image EcoRI restriction ... enzyme EcoRI Eco RI cyan and green cartoon diagram bound to double stranded DNA brown tubes . ref ... text align center background beige colspan 3 Derivation of the EcoRI name Abbreviation Meaning ... pmid 12654995 pmc 152790 doi 10.1093 nar gkg274 url ref For example, the name of the EcoRI restriction ... solid aaa border collapse collapse Enzyme Source Recognition Sequence Cut EcoRI Escherichia coli 5 ... EcoRI , HindIII , BglII . List of restriction enzyme cutting sites Homing endonuclease List of homing ... more details
File pBR322.svg thumb The vector pBR322 with exemplified restriction sites. pBR322 is a plasmid and was the first widely used E. coli cloning Vector molecular biology vector s. Created in 1977 in the laboratory of Herbert Boyer at the University of California San Francisco, it was named after the Mexican postdoctoral researchers who constructed it. The p stands for plasmid, and BR for Bolivar and Rodriguez. pBR322 is 4361 base pairs ref cite journal author Watson, N. title A new revision of the sequence of plasmid pBR322 journal Gene volume 70 pages 399 403 year 1988 pmid 3063608 doi 10.1016 0378 1119 88 90212 0 issue 2 ref in length and contains the replicon of plasmid pMB1, the amp sup R sup gene, encoding the ampicillin Antibiotic resistance resistance protein source plasmid RSF2124 and the tet sup R sup gene, encoding the tetracycline resistance protein source plasmid pSC101 . The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tet sup R sup gene. There are 2 sites for restriction enzymes HindIII and ClaI within the promoter of the tet sup R sup gene. There are 6 key restriction sites inside the amp sup R sup gene. The origin of replication or Ori genetics ori site in this plasmid is pMB1 a close relative of ColE1 . ref cite web url http www1.qiagen.com Plasmid BacterialCultures.aspx tab2 title Growth of Bacterial Cultures author Qiagen Plasmid Resource Center ref The ori encodes two RNAs RNAI and RNAII and one protein called Rom or Rop protein Rop . The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet gene. The ampicillin resistance gene is penicillin beta lactamase. Promoters P1 and P3 are for the beta lactamase gene. P3 is the natural promoter, and P1 is artificially created by the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates Transcr ... more details
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