Image EcoRI restriction enzyme recognition site.svg thumb 175px EcoRI recognition site with cutting pattern indicated by a green line Image ECOR1 Crystal Structure.rsh.png thumb EcoRI crystal structure EcoRI pronounced eco R one 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, D91, E111, A112, K113 2 1. Tertiary and Quaternary Structure The enzyme is a homodimer of a 31 kilodalton subunit consisting of one globular domain of the architecture. Each subunit contains a loop which sticks out from the globular domain and wraps around the DNA when bound 3 . EcoRI has been cocrystallized with the sequence it normally cuts. This crystal was used to solve the structure http www.pdb.org pdb explore.do?structureId 1QPS of the complex ... to communicate 4 . Uses Restriction enzymes such as EcoRI are used in a wide variety of molecular genetics ... . Restriction enzymes like EcoRI that generate sticky ends of DNA are often used to cut DNA prior to ligation, as the sticky ends make the ligation reaction more efficient. EcoRI can exhibit non .... Conditions 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 Eco RI he EcoRI ja EcoRI pl EcoRI sv EcoRI zh EcoRI ... more details
Unreferenced date December 2009 Restriction sites , or restriction recognition sites , are locations on a DNA molecule containing specific sequences of nucleotide s, which are recognized by restriction enzyme s. These are generally palindromic sequence s 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 C on both the top and bottom strands, leaving an overhang an end portion of a DNA strand with no attached complement 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 . References DEFAULTSORT Restriction Site Category Molecular biology fr Sites de restriction it Sito di restrizione ru ... more details
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. date 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 cite book title Introduction to Biotechnology author Thieman, W.J. and Palladino, M.A. date 2004 publisher Pearson Education, Benjamin Cummings page 55 ref . The cloning of genes into plasmids occurred soon after. In 1980 ref name Introduction to Biotechnology cite book title Introduction to Biotechnology author Thieman, W.J. and Palladino, M.A. date 2004 publisher Pearson Education, Benjamin Cummings page 55 ref , 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
orientation. In this example the orientation of an insert cloned with EcoRI will be found. Digests 1 EcoRI 2 HindIII 3 EcoRI HindIII optional and not discussed Resultant Fragments approximate ... 5 HindIII EcoRI 3 Discussion The EcoRI digest excises the insert yielding fragments of 3 kb and 5 ... 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 5 GAATTC 3 CTTAAG 5 G AATTC 3 3 CTTAA G ... Enzyme Action of EcoRI http www.accessexcellence.org AE AEC CC enzyme glossary.html Enzyme glossary ... 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 canonical 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 , for example, star activity in HindIII 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 ... more details
common and has greater biological importance than the mirror like. EcoRI digestion produces sticky ends, Image EcoRI restriction enzyme recognition site.svg 90px whereas SmaI restriction enzyme ... 240px right Structure of the protein dimer homodimeric restriction enzyme EcoRI Eco RI cyan and green ... align center background beige colspan 3 Derivation of the EcoRI name Abbreviation Meaning Description ... doi 10.1093 nar gkg274 url ref For example, the name of the EcoRI restriction enzyme was derived as shown ... collapse Enzyme Source Recognition Sequence Cut EcoRI Escherichia coli 5 GAATTC 3 CTTAAG 5 G AATTC ... about certain restriction enzymes EcoRI , HindIII , BglII . List of restriction enzyme cutting ... more details
Escherichia coli and, later, the EcoRI derivative, ref name isbn0 1218 1968 X cite book author ... by the pBR322 plasmid. ref name isbn0 1218 1968 X In the case of EcoRI, the plasmid can anneal ... more details
File PBR322.svg thumb The vector pBR322 with exemplified restriction sites. pBR322 is a plasmid and for a time was one of the most commonly used E. coli cloning Vector molecular biology vector s. Created in 1977, it was named eponymously after its Mexican creators, p standing 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 accessdate 2009 03 27 pmid 3063608 doi 10.1016 0378 1119 88 90212 0 issue 2 ref in length and contains a replicon region source 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 . The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet genes. The ampicillin resistance gene is a 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 Transcription genetics transcription in the direction of the tetracycli ... more details
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