Chapter 2 Enzymes Used in GE

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Chapter 2 Enzymes used in GE Ø2.1 Restriction endonuclease Ø2.2 DNA ligase joining DNA molecules Ø2.3 DNA Polymerase and modifying enzymes Ø2.4 Nucleases 2.1 Restriction endonuclease • 1952年，Luria和Human，T偶数噬菌体 • 1953年，Bertani和Weigle，λ噬菌体 Restriction endonuclease: recognizes a short and specific DNA sequence and cuts it from inside. The specific DNA sequence is called recognition sequence cleavage site：cut site How restriction enzymes are named? Escherichia Coli Ry13 EcoR I 属名种名株系编号若种名头2个字母相同则其中一个可用种名的第一和第三个字母。书写时前3个字母一定斜体，字母与罗马数字间要有空格。 Classification Recognition Sequences • Many restriction sequences are palindromic(回文,Read the same in the opposite direction ). For example, –5’ GAATTC 3’ –3’ CTTAAG 5’ • 回文诗：春城一色柳垂新，色柳垂新自爱人人爱自新垂柳色，新垂柳色一城春 Sticky End Cutters • Staggered cuts produce single stranded “sticky-ends” • A protruding, single-stranded nucleotide sequence produced when a restriction endonuclease cleaves off center in its recognition sequence. Blunt End Cutters • cutting across both strands of DNA. • These are called blunt end cutters AluI HaeIII isoschizomers.(同裂酶) • Restriction enzymes that have the same recognition sequence as well as the same cleavage site . Neoschizomers (新裂酶) • Some restriction enzymes cut the same recognition site but at different locations –Neoschizomers are a subset of isoschizomers. isocaudomers同尾酶 • Some restriction endonucleases produce the same nucleotide extensions but have different recognition sites complete digestion完全的酶切: cleave at all relevant restriction sites. partial digestion局部酶切: a restriction enzyme is controlled so that it cuts only some of the available sites. Storage • storage at –20°C for most restriction enzymes. • for periods longer than 30 days, storage at –70°C. • Glycerol(50%） is added to restriction enzyme storage buffers to prevent freezing at -20°C. • 10X Buffers and concentrated BSA should also be stored at –20°C. Digestion Assay restriction endonuclease activity: One unit of is defined the amount of enzyme required to completely digest 1 μg of substrate DNA ( λDNA） in a total reaction volume of 50 μl in an hour. Volume activity: U/μl.

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Restriction Endonucleases
Restriction Endonucleases TECHNICAL GUIDE UPDATE 2017/18 be INSPIRED drive DISCOVERY stay GENUINE RESTRICTION ENZYMES FROM NEB Cut Smarter with Restriction Enzymes from NEB® Looking to bring CONVENIENCE to your workflow? Simplify Reaction Setup and Double Activity of DNA Modifying Enzymes in CutSmart Buffer: Digestion with CutSmart® Buffer Clone Smarter! Activity Enzyme Required Supplements Over 210 restriction enzymes are 100% active in a single buffer, in CutSmart Phosphatases: CutSmart Buffer, making it significantly easier to set up your Alkaline Phosphatase (CIP) + + + double digest reactions. Since CutSmart Buffer includes BSA, there Antarctic Phosphatase + + + Requires Zn2+ Quick CIP + + + are fewer tubes and pipetting steps to worry about. Additionally, Shrimp Alkaline Phosphatase (rSAP) + + + many DNA modifying enzymes are 100% active in CutSmart Ligases: T4 DNA Ligase + + + Requires ATP Buffer, eliminating the need for subsequent purification. E. coli DNA Ligase + + + Requires NAD T3 DNA Ligase + + + Requires ATP + PEG For more information, visit www.NEBCutSmart.com T7 DNA Ligase + + + Requires ATP + PEG Polymerases: T4 DNA Polymerase + + + DNA Polymerase I, Large (Klenow) Frag. + + + DNA Polymerase I + + + DNA Polymerase Klenow Exo– + + + Bst DNA Polymerase + + + ™ phi29 DNA Polymerase + + + Speed up Digestions with Time-Saver T7 DNA Polymerase (unmodified) + + + Qualified Restriction Enzymes Transferases/Kinases: T4 Polynucleotide Kinase + + + Requires ATP + DTT T4 PNK (3´ phosphatase minus) + + + Requires ATP + DTT > 190 of our restriction enzymes are able to digest DNA in CpG Methyltransferase (M. SssI) + + + 5–15 minutes, and can safely be used overnight with no loss of GpC Methyltransferase (M. CviPI) + Requires DTT T4 Phage β-glucosyltransferase + + + sample. For added convenience and flexibility, most of these are Nucleases, other: supplied with CutSmart Buffer.
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Gel Electrophoresis Applications Gel Electrophoresis Applications
Restriction Enzymes (also known as restriction endonucleases) What are restriction enzymes? • An enzyme that cuts DNA at specific nucleotide sequences known as restriction sites • Naturally found in bacteria and have evolved to provide a defense mechanism against invading viruses • In bacteria, restriction enzymes selectively cut up foreign DNA in a process called restriction Restriction Enzymes in Bacteria Restriction Enzymes • Cut both strands of the DNA double helix • Over 3000 enzymes have been identified • More than 600 available commercially • Routinely used for DNA modification and manipulation in laboratories (“molecular scissors”) Restriction Sites • Also known as recognition sites • Generally genetic palindromic sequences • A palindromic sequence in DNA one in which the 5’ to 3’ base pair sequence is identical on both strands • Usually a 4 or 6 base pair sequence Restriction Sites • The enzymes scan DNA sequences, find a very specific set of nucleotides and make a cut Hae III • HaeIII is a restriction enzyme that searches the DNA molecule until it finds this sequence of four nitrogen bases - GGCC 5’ TGACGGGTTCGAGGCCAG 3’ 3’ ACTGCCCAAGGTCCGGTC 5’ Hae III • Once the recognition site was found HaeIII will go to work cutting (cleaving) the DNA Blunt Ends versus Sticky Ends • Hae III produces “blunt ends” when cleaving DNA • Other enzymes produce “sticky ends” blunt end sticky end Restriction Enzyme Names • Named after the type of bacteria in which the enzyme is found and the order in which the restriction enzyme was identified and isolated EcoRI for example R strain of E.coli bacteria I as it is was the first E. coli restriction enzyme to be discovered.
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Specific Cleavageanalysis of Mammalian Mitochondrial
Proc. Nat. Acad. Sci. USA Vol. 72, No. 11, pp. 4496-4500, November 1975 Cell Biology Specific cleavage analysis of mammalian mitochondrial DNA (restriction endonuclease/gel electrophoresis/interspecies comparisons/intraspecies differences) S. STEVEN POTTER, JOHN E. NEWBOLD, CLYDE A. HUTCHISON III, AND MARSHALL H. EDGELL Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, N.C. 27514 Communicated by Jerome Vinograd, September 5, 1975 ABSTRACT Mitochondrial DNA from several mamma- fresh from local slaughterhouses; dog, rabbit, rat, and ham- lian species has been digested with a site-specific restriction ster (Syrian golden) specimens were obtained from conven- endonuclease (HaeIII) from Haemophilus aegyptius. A quan- titative analysis of the resulting specific fragments indicates tional suppliers, and sacrificed for fresh tissues in the labora- that the mtDNA of any individual mammal is predominantly tory; mice were obtained from Dr. G. Haughton; African a single molecular clone. green monkey livers were purchased from Flow Laborato- Gel analysis of specific cleavage products has proven quite ries; chimpanzee livers were provided by Dr. R. Metzgar of sensitive in detecting differences in mtDNA: mtDNAs from Duke University; buffalo (Bos bison) liver was obtained the more distantly related mammals studied (e.g., donkey from the Buffalo Ranch, Concord, N.C.; human hearts and and dog) are found to have few bands in common, and very closely related mammals (e.g., donkey and horse) share only livers were obtained as autopsy samples from the N.C. Me- about 50% of their bands. This procedure has detected sever- morial Hospital, Chapel Hill; leukocytes (removed as part of al intraspecies mtDNA differences.
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Identification and Characterization of Cbei, a Novel Thermostable
J Ind Microbiol Biotechnol DOI 10.1007/s10295-011-0976-x ORIGINAL PAPER Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes Dae-Hwan Chung • Jennifer R. Huddleston • Joel Farkas • Janet Westpheling Received: 27 January 2011 / Accepted: 7 April 2011 Ó Society for Industrial Microbiology 2011 Abstract Potent HaeIII-like DNA restriction activity was Keywords Caldicellulosiruptor Á Cellulolytic Á detected in cell-free extracts of Caldicellulosiruptor bescii Thermophile Á Anaerobe Á HaeIII Á Restriction-modification DSM 6725 using plasmid DNA isolated from Escherichia system Á Thermostable restriction enzyme coli as substrate. Incubation of the plasmid DNA in vitro with HaeIII methyltransferase protected it from cleavage by HaeIII nuclease as well as cell-free extracts of C. bescii. Introduction The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His-tag at the Caldicellulosiruptor bescii DSM 6725 (formerly Anaero- C-terminus. The purified protein was 38 kDa as predicted cellum thermophilum [31]) grows at up to 90°C and is the by the 981-bp nucleic acid sequence, was optimally active most thermophilic cellulolytic bacterium known. This at temperatures between 75°C and 85°C, and was stable for obligate anaerobe is capable of degrading lignocellulosic more than 1 week when stored at 35°C. The cleavage biomass including hardwood (poplar) and grasses with both sequence was determined to be 50-GG/CC-30, indicating low lignin (Napier grass, Bermuda grass) and high lignin that CbeI is an isoschizomer of HaeIII.
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PG 4Th Semester
Paper No. : 04 Genetic engineering and recombinant DNA technology Module : 01 History of Genetic Material Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi Paper Coordinator: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi Content Writer: Dr Rohini Muthuswami, Associate Professor, Jawaharlal Nehru University Content Reviwer: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi Genetic Engineering and Recombinant DNA Technology Biotechnology History of Genetic Material Description of Module Subject Name Biotechnology Paper Name Genetic Engineering and Recombinant DNA Technology Module Name/Title History of Genetic Material Module Id 01 Pre-requisites Objectives To understand how DNA was identified as genetic material Keywords DNA Genetic Engineering and Recombinant DNA Technology Biotechnology History of Genetic Material 1. Nature of the genetic material. In this module we will learn about how scientists discovered the identity of the genetic material. The foundation of modern biology was laid by scientists in mid-19th century. The principles of genetics were discovered by Gregor Mendel in 1865-66. The nuclein or what we term as nucleic acid was discovered and identified in 1869. The final identity of the genetic material was firmly established in 1952, two years after the structure of DNA was elucidated. We are going to follow this fascinating journey and I hope at the end of this module you learn to appreciate how scientists using the simple tools and techniques available to them established the identity of the genetic material.
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Site-Specific Cleavage of Single-Stranded DNA by A
Proc. Nat. Acad. Sci. USA VoL 72, No. 7, pp. 2555-2558, July 1975 Biochemistry Site-specific cleavage of single-stranded DNA by a Hemophilus' restriction endonuclease (fl phage DNA/endo R-HaeIII/fragments of single strands) KENSUKE HORIUCHI AND NORTON D. ZINDER The Rockefeller University, New York, N.Y. 10021 Contributed by Norton D. Zinder, April 15,1975 ABSTRACT Single-stranded viral DNA of bacteriophage E. coli strain used for the preparation of RFI, was very gen- fI is cleaved into specific fragments by endo R-HaeIII, a re- erously provided by Dr. S. Hattman (23). Preparation of striction endonuclease isolated from Hemophilus aegyptius. 32P-labeled DNA has been described (12). The sites of the single strand cleavage correspond to those of the double strand cleavage. A single-stranded DNA fragment Endonucleases. Endonucleases R.HaeIII (6, 12), R-HaeII containing only one HaeIII site is also cleaved by this en- (11, 12), and R-Hind (4) were isolated as described previous- zyme. This observation suggests that the reaction of single- ly (12). Endo R-EcoRII, isolated by the method described by stranded DNA cleavage does not require the formation of a Yoshimori (5), was supplied by Dr. G. F. Vovis. symmetrical double-stranded structure that would result Denaturation and Renaturation. Alkali denaturation and from the intramolecular base-pairing between two different subsequent renaturation of DNA were carried out as de- HaeIII sites. Other restriction endonucleases may also cleave scribed previously (24) except that acetic acid was used in- single-stranded DNA. stead of NaH2PO4 to neutralize the alkali. Various restriction endonucleases have been isolated which Gel Electrophoresis.
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Overcoming Restriction As a Barrier to DNA Transformation In
Chung et al. Biotechnology for Biofuels 2013, 6:82 http://www.biotechnologyforbiofuels.com/content/6/1/82 RESEARCH Open Access Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement Daehwan Chung1,2, Joel Farkas1,2 and Janet Westpheling1,2* Abstract Background: Thermophilic microorganisms have special advantages for the conversion of plant biomass to fuels and chemicals. Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria known. They have the ability to grow on a variety of non-pretreated biomass substrates at or near ~80°C and hold promise for converting biomass to bioproducts in a single step. As for all such relatively uncharacterized organisms with desirable traits, the ability to genetically manipulate them is a prerequisite for making them useful. Metabolic engineering of pathways for product synthesis is relatively simple compared to engineering the ability to utilize non-pretreated biomass. Results: Here we report the construction of a deletion of cbeI (Cbes2438), which encodes a restriction endonuclease that is as a major barrier to DNA transformation of C. bescii. This is the first example of a targeted chromosomal deletion generated by homologous recombination in this genus and the resulting mutant, JWCB018 (ΔpyrFA ΔcbeI), is readily transformed by DNA isolated from E. coli without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (Cbes2437) intact. This was confirmed by the fact that DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII. Plasmid DNA isolated from C. hydrothermalis transformants were readily transformed into C.
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1.Ptii.Snps and TAS2R38.V3
Using Single Nucleotide Polymorphism (SNP) to Predict Bitter Tasting Ability Part II: ! Digestion and Analysis of an Amplified Region of the Bitter Taste Receptor TAS2R38 Gene In The Last Lab: •! You sampled some of your own DNA and amplified a short region of chromosome 7. •! This region codes for part of TAS2R38 gene, bitter taste receptor gene. In Today’s Lab: •! You will digest some of your amplified PCR product with the restriction enzyme, Hae III •! And use agarose gel electrophoresis to determine your genotype •! You will also taste a sample of PTC paper and determine your phenotype •! You will compare the results of your genotype with those of your phenotype – record on board •! Finally, you will use class data to determine if the class is in Hardy-Weinberg equilibrium Digestion of TAS2R38 With Restriction Enzyme, Hae III Agarose Gel Electrophoresis Digesting PCR Sample with HAE III Get your PCR tube from your instructor or TA (need your code) •! Transfer 16 µl (0.016 ml) of your PCR product (amplified DNA) to a flat top PCR tube •! Label this tube “D” for “digested” and add your code. •! This DNA sample will be digested with the restriction enzyme, HaeIII •! Ask your instructor or TA to add 1µl (0.001 ml) of the restriction enzyme, HaeIII, to the “D” tube •! Place this tube in the thermal cycler set for 37oC and incubate for at least 30 minutes. Label your original PCR tube with a “U” for undigested; leave this sample on ice while the other sample is incubated with HaeIII. ! http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf
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The Lspc3–41I Restriction-Modification System Is
Fu et al. BMC Microbiology (2017) 17:116 DOI 10.1186/s12866-017-1014-6 RESEARCH ARTICLE Open Access The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41 Pan Fu1,2, Yong Ge1, Yiming Wu1, Ni Zhao1, Zhiming Yuan1* and Xiaomin Hu1* Abstract Background: Lysinibacillus sphaericus has been widely used in integrated mosquito control program and it is one of the minority bacterial species unable to metabolize carbohydrates. In consideration of the high genetic conservation at genomic level and difficulty of genetic horizontal transfer, it is hypothesized that effective restriction-modification (R-M) systems existed in mosquitocidal L. sphaericus. Results: In this study, six type II R-M systems including LspC3–41I were predicted in L. sphaericus C3–41 genome. It was found that the cell free extracts (CFE) from this strain shown similar restriction and methylation activity on exogenous Bacillus/Escherichia coli shuttle vector pBU4 as the HaeIII, which is an isoschizomer of BspRI. The Bsph_0498 (encoding the predicted LspC3–41IR) knockout mutant Δ0498 and the complement strain RC0498 were constructed. It was found that the unmethylated pBU4 can be digested by the CFE of C3–41 and RC0498, but not by that of Δ0498. Furthermore, the exogenous plasmid pBU4 can be transformed at very high efficacy into Δ0498, low efficacy into RC0498, but no transformation into C3–41, indicating that LspC3–41I might be a major determinant for the genetic restriction barrier of strain C3–41. Besides, lspC3–41IR and lspC3–41IM genes are detected in other two strains besides C3–41 of the tested 16 L.
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REBASE — Restriction Enzymes and Methylases
3628-3639 Nucleic Acids Research, 1994, Vol. 22, No. 17 .:j 1994 Oxford University Press REBASE restriction enzymes and methylases Richard J.Roberts* and Dana Macelis New England BioLabs, 32 Tozer Road, Beverly, MA 01915, USA ABSTRACT REBASE is a comprehensive database of information a monthly release note indicating that the files at the ftp site have about restriction enzymes and their associated been updated, and listing new enzymes, newly available formats, methylases, including their recognition and cleavage enzyme name changes, etc. To join the mailing list or for more sites and their commercial availability. Information from information, send a request to R.J.Roberts via e-mail to REBASE is available via monthly electronic mailings as [email protected], telephone (508)927-3382 or Fax (508)921- well as via WAIS and anonymous ftp. Specialized files 1527. It should be noted that REBASE is now accessible via are available that can be used directly by many software WAIS directly from vent.neb.com (192.138.220.2 port 210). We packages. currently have five WAIS sources set up. REBASE_help: general description of REBASE and the services and data files offered. INTRODUCTION REBASE-enzymes: facts about each enzyme in REBASE. The restriction enzyme database, REBASE, is a collection of REBASE-references: all the published references stored in information about restriction enzymes and methylases. Since the REBASE complete with abstracts where available. last description of the contents of REBASE (1), 138 new entries REBASE-suppliers: commmercial sources of enzymes, includes have been added including 10 new Type II enzymes: AceIH, C- contact information (address, telephone nos, fax nos) for each AGCTC (7/11); BmgI, GKGCCC; BsbI, CAACAC;.
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Restriction Endonucleases
Restriction Endonucleases TECHNICAL GUIDE UPDATE 2017/18 be INSPIRED drive DISCOVERY stay GENUINE RESTRICTION ENZYMES FROM NEB Cut Smarter with Restriction Enzymes from NEB® Looking to bring CONVENIENCE to your workflow? Simplify Reaction Setup and Double Activity of DNA Modifying Enzymes in CutSmart Buffer: Digestion with CutSmart® Buffer Clone Smarter! Activity Enzyme Required Supplements Over 210 restriction enzymes are 100% active in a single buffer, in CutSmart Phosphatases: CutSmart Buffer, making it significantly easier to set up your Alkaline Phosphatase (CIP) + + + double digest reactions. Since CutSmart Buffer includes BSA, there Antarctic Phosphatase + + + Requires Zn2+ Quick CIP + + + are fewer tubes and pipetting steps to worry about. Additionally, Shrimp Alkaline Phosphatase (rSAP) + + + many DNA modifying enzymes are 100% active in CutSmart Ligases: T4 DNA Ligase + + + Requires ATP Buffer, eliminating the need for subsequent purification. E. coli DNA Ligase + + + Requires NAD T3 DNA Ligase + + + Requires ATP + PEG For more information, visit www.NEBCutSmart.com T7 DNA Ligase + + + Requires ATP + PEG Polymerases: T4 DNA Polymerase + + + DNA Polymerase I, Large (Klenow) Frag. + + + DNA Polymerase I + + + DNA Polymerase Klenow Exo– + + + Bst DNA Polymerase + + + ™ phi29 DNA Polymerase + + + Speed up Digestions with Time-Saver T7 DNA Polymerase (unmodified) + + + Qualified Restriction Enzymes Transferases/Kinases: T4 Polynucleotide Kinase + + + Requires ATP + DTT T4 PNK (3´ phosphatase minus) + + + Requires ATP + DTT > 190 of our restriction enzymes are able to digest DNA in CpG Methyltransferase (M. SssI) + + + 5–15 minutes, and can safely be used overnight with no loss of GpC Methyltransferase (M. CviPI) + Requires DTT T4 Phage β-glucosyltransferase + + + sample. For added convenience and flexibility, most of these are Nucleases, other: supplied with CutSmart Buffer.
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Restriction Endonucleases, Isoschizomers
Paper No. : 04 Genetic engineering and Recombinant DNA technology Module : 03 Restriction Endonucleases, Isoschizomers Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi Course Coordinator: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia Content Writer: Dr. Pravir Kumar, Professor, Department of Biotechnology, Delhi Technological University (Formerly Delhi College of Engineering) Content Reviwer: Dr Rohini Muthuswami, Associate Professor, Jawaharlal Nehru University Genetic engineering and Recombinant DNA technology Biotechnology Restriction Endonucleases, Isoschizomers Description of Module Subject Name Biotechnology Paper Name Genetic engineering and recombinant DNA technology Module Name/Title Restriction Endonucleases, Isoschizomers Module Id 03 Pre-requisites Objectives Keywords Genetic engineering and Recombinant DNA technology Biotechnology Restriction Endonucleases, Isoschizomers INTRODUCTION ABOUT RESTRICTION ENZYMES Gene manipulation is a revolutionary step to understand gene function and its proper usage to correct DNA sequence in a desired fashion. Gene cloning is one such method by which we can introduce gene of interest in a suitable vector system and then introduced that construct in an appropriate cell system to check the yield of manipulated gene sequence. In order to join or cut the large DNA chunk, we need
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