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Similarly , cutting DNA and pasting DNA fragments together typically are among the first techniques learned in the molecular biology lab and are fundamental to all recombinant DNA work.Such manipulations of DNA are conducted by a toolkit of enzymes:: Such manipulations of DNA are conducted by a toolkit of enzymes: restriction endonucleases are used as molecular scissors, DNA ligase functions to bond pieces of DNA together, and a variety of additional enzymes that modify DNA are used to facilitate the process.DNA modifying enzymes: DNA modifying enzymes Restriction enzymes and DNA ligases represent the cutting and joining functions in DNA manipulation. All other enzymes involved in genetic engineering fall under the broad category of enzymes known as DNA modifying enzymes. These enzymes are involved in the degradation, synthesis and alteration of the nucleic acids.PowerPoint Presentation: Types Of Modifying EnzymesNucleases: Nucleases Nuclease enzymes degrade nucleic acids by breaking the phosphodiester bond that holds the nucleotides together. Restriction enzymes are good examples of endonucleases, which cut within a DNA strand. A second group of nucleases, which degrade DNA from the termini of the molecule, are known as exonucleases .PowerPoint Presentation: Apart from restriction enzymes, there are four useful nucleases that are often used in genetic engineering. These are Bal 31 and exonuclease III ( exonucleases ), and deoxyribonuclease I ( DNase I) and S1-nuclease (endonucleases ). These enzymes differ in their precise mode of action and provide the genetic engineer with a variety of strategies for attacking DNA. Their features are summarised in Fig. (next slide)Nucleases and its action: N ucleases and its action Fig.,Mode of action of various nucleases.: Mode of action of various nucleases. Nuclease Bal 31 is a complex enzyme. Its primary activity is a fast-acting 3’ exonuclease , which is coupled with a slow-acting endonuclease. When Bal 31 is present at a high concentration these activities effectively shorten DNA molecules from both termini . Exonuclease III is a 3’ exonuclease that generates molecules with protruding 5’ termini. DNase I cuts either single-stranded or double-stranded DNA at essentially random sites. Nuclease S1 is specific for single-stranded RNA or DNA .PowerPoint Presentation: In addition to DNA-specific nucleases, there are ribonucleases ( RNases ), which act on RNA. These may be required for many of the stages in the preparation and analysis of recombinants and are usually used to get rid of unwanted RNA in the preparation. However, as well as being useful, ribonucleases can pose some unwanted problems. They are remarkably difficult to inactivate and can be secreted in sweat.PowerPoint Presentation: Thus, contamination with RNases can be a problem in preparing recombinant DNA, particularly where cDNA is prepared from an mRNA template. In this case it is vital to avoid RNase contamination by wearing gloves and ensuring that all glass and plastic equipment is treated to avoid ribonuclease contamination. Not all nucleases are helpful! Ribonucleases can be a problem when working with purified preparations of RNA, and care must be taken to remove or inactivate RNase activity.Polymerases: Polymerases Polymerase enzymes synthesise copies of nucleic acid molecules and are used in many genetic engineering procedures. When describing a polymerase enzyme, the terms ‘DNA-dependent’ or ‘ RNA-dependent’ may be used to indicate the type of nucleic acid template that the enzyme uses. Thus , a DNA-dependent DNA polymerase copies DNA into DNA, an RNA-dependent DNA polymerase copies RNA into DNA , and a DNA-dependent RNA polymerase transcribes DNA into RNA .PowerPoint Presentation: These enzymes synthesise nucleic acids by joining together nucleotides whose bases are complementary to the template strand bases. The synthesis proceeds in a 5’→3’ direction, as each subsequent nucleotide addition requires a free 3’-OH group for the formation of the phosphodiester bond. This requirement also means that a short double-stranded region with an exposed 3’-OH (a primer) is necessary for synthesis to begin.PowerPoint Presentation: Polymerases are the copying enzymes of the cell; they are also essential parts of the genetic engineer’s armoury. These enzymes are template-dependent and can be used to copy long stretches of DNA or RNA.PowerPoint Presentation: The enzyme DNA polymerase I has, in addition to its polymerase function, 5’→3’ and 3’→5’ exonuclease activities. The enzyme catalyses a strand-replacement reaction, where the 5’→3’ exonuclease function degrades the non-template strand as the polymerase synthesises the new copy. A major use of this enzyme is in the nick translation procedure for radiolabelling DNA. The 5’→3’ exonuclease function of DNA polymerase I can be removed by cleaving the enzyme to produce what is known as the Klenow fragment .PowerPoint Presentation: This retains the polymerase and 3’→5’ exonuclease activities. The Klenow fragment is used where a single-stranded DNA molecule needs to be copied; because the 5’→3’ exonuclease function is missing, the enzyme cannot degrade the non-template strand of dsDNA during synthesis of the new DNA. The 3’→5’ exonuclease activity is supressed under the conditions normally used for the reaction .PowerPoint Presentation: Major uses for the Klenow fragment include radiolabelling by primed synthesis and DNA sequencing by the dideoxy method in addition to the copying of single-stranded DNAs during the production of recombinants. A modified form of DNA polymerase I called the Klenow fragment is a useful polymerase that is used widely in a number of applications .PowerPoint Presentation: Reverse transcriptase ( RTase ) is an RNA-dependent DNA polymerase, and therefore produces a DNA strand from an RNA template. It has no associated exonuclease activity. The enzyme is used mainly for copying mRNA molecules in the preparation of cDNA ( complementary or copy DNA ) for cloning, although it will also act on DNA templates . Reverse transcriptase is a key enzyme in the generation of cDNA ; the enzyme is an RNA-dependent DNA polymerase, which produces a DNA copy of an mRNA molecule.Enzymes that modify the ends of DNA molecules: Enzymes that modify the ends of DNA molecules The enzymes alkaline phosphatase, polynucleotide kinase, and terminal transferase act on the termini of DNA molecules and provide important functions that are used in a variety of ways. The phosphatase and kinase enzymes, as their names suggest, are involved in the removal or addition of phosphate groups. Bacterial alkaline phosphatase (there is also a similar enzyme, calf intestinal alkaline phosphatase) removes phosphate groups from the 5 ends of DNA, leaving a 5-OH group.PowerPoint Presentation: The enzyme is used to prevent unwanted ligation of DNA molecules, which can be a problem in certain cloning procedures. It is also used prior to the addition of radioactive phosphate to the 5 ends of DNAs by polynucleotide kinase. Terminal transferase (terminal deoxynucleotidyl transferase) repeatedly adds nucleotides to any available 3 terminus.PowerPoint Presentation: Although it works best on protruding 3 ends, conditions can be adjusted so that blunt-ended or 3-recessed molecules may be utilised. The enzyme is mainly used to add homopolymer tails to DNA molecules prior to the construction of recombinants. In many applications it is often necessary to modify the ends of DNA molecules using enzymes such as phosphatases, kinases, and transferases .DNA Polymerases: DNA Polymerases Mesophilic and thermophilic DNA polymerases for different polymerization reactions, DNA end blunting and amplification, labeling and others. DNA Polymerase, Large Fragment DNA Polymerase I Klenow Fragment Klenow Fragment, exo – phi29 DNA Polymerase T4 DNA Polymerase T7 DNA Polymerase Terminal Deoxynucleotidyl Transferase Terminal Transferase ( TdT )DNA Polymerase, Large Fragment: DNA Polymerase , Large Fragment DNA Polymerase, Large Fragment, is a portion of DNA polymerase of Bacillus smithii , which catalyzes 5'=>3' synthesis of DNA and lacks 5'→3' and 3'→5' exonuclease activities. DNA Polymerase, Large Fragment, has a strong strand displacement activity and is active in a wide range of temperatures from 30°C to 63°C, with an optimum of activity at 60° . It is an enzyme with high functional similarity to DNA Polymerase, Large Fragment, and can replace it in most applications. Not suitable for use in PCR .PowerPoint Presentation: Highlights Thermophilic DNA polymerase with strong strand displacement activity Use of this enzyme in certain applications may be covered by patents and may require a license.PowerPoint Presentation: Applications Isothermal DNA amplification by the method of: Loop-mediated isothermal amplification ( LAMP) Whole genome amplification (WGA) Ramification amplification (RAM) Random-primed DNA labeling Labeling by fill-in 5'-overhangs of dsDNADNA Polymerase I: DNA Polymerase I DNA Polymerase I, a template-dependent DNA polymerase, catalyzes 5'→3' synthesis of DNA. The enzyme also exhibits 3'→5' exonuclease (proofreading) activity, 5'→3' exonuclease activity, and ribonuclease H activity. Highlights Incorporates modified nucleotides (e.g. biotin-, digoxigenin -, aminoallyl -, fluorescently- labeled nucleotides) Active in multiple buffers , including restriction enzyme, PCR, and RT buffers Applications DNA labeling by nick-translation in conjunction with Dnase Second-strand synthesis of cDNA in conjunction with RNaseHPowerPoint Presentation: Inactivation Inactivated by heating at 75°C for 10 min or by addition of EDTA. Inhibition Inhibitors: metal chelators, PP i , P i (at high concentrations) (see Reference 5). Molecular Weight 103 kDa monomer Quality Control The absence of endodeoxyribonucleases confirmed by appropriate quality test. Source E.coli cells with a cloned polA gene.Klenow Fragment: Klenow Fragment Klenow Fragment is the large fragment of DNA polymerase I. It exhibits 5'→3' polymerase activity and 3'→5' exonuclease (proofreading) activity, but lacks 5'→3' exonuclease activity of DNA polymerase I . Highlights Incorporates modified nucleotides (e.g., Cy3-, Cy5-, aminoallyl -, biotin-, digoxigenin - and fluorescently- labeled nucleotides) Active in restriction enzyme, PCR, RT, and T4 DNA Ligase buffersPowerPoint Presentation: Applications DNA blunting by fill-in 5'- overhangs Random-primed DNA labeling Labeling by fill-in 5'-overhangs of dsDNA DNA sequencing by the Sanger method Site-specific mutagenesis of DNA with synthetic oligonucleotides Second strand synthesis of cDNASynthesis of double-stranded DNA from single-stranded templates:: Synthesis of double-stranded DNA from single-stranded templates :Filling in recessed 3' ends of DNA fragments:: Filling in recessed 3' ends of DNA fragments :Digesting away protruding 3' overhangs:: Digesting away protruding 3' overhangs :PowerPoint Presentation: Inactivation Inactivated by heating at 75°C for 10min or by addition of EDTA. Inhibition Inhibitors: metal chelators, PPi, Pi (at high concentrations) (see Reference 8). Molecular Weight 68kDa monomer. Quality Control The absence of endodeoxyribonucleases confirmed by appropriate quality test. Functionally tested for fill in of 5'-overhanging DNA termini and for random primed DNA labeling . Source E. coli cells with a cloned fragment of the polA gene.phi29 DNA Polymerase: phi29 DNA Polymerase phi29 DNA Polymerase is a highly processive polymerase (up to more than 70 kb) featuring strong strand displacement activity, which allows for highly efficient isothermal DNA amplification. phi29 DNA Polymerase also possesses a 3'→5' exonuclease (proofreading) activity acting preferentially on single-stranded DNA or RNA. Therefore 3'-modified primers are highly recommended. Addition of Pyrophosphatase to the reaction mixture with phi29 DNA Polymerase may enhance DNA synthesis.PowerPoint Presentation: Highlights Highest processivity and strand displacement activity among known DNA polymerases – more than 70 kb long DNA stretches can be synthesized. Highly accurate DNA synthesis . Extremely high yields of amplified DNA even from minute amounts of template Amplification products can be directly used in downstream applications (PCR, restriction digestion, SNP genotyping, etc.)PowerPoint Presentation: Applications Rolling circle amplification (RCA ) : generation of periodic DNA nanotemplates Multiple displacement amplification (MDA) Unbiased amplification of whole genome ( WGA): amplification of DNA for SNP and STR detection cell-free amplification of DNA from single cells pathogenic organisms or metagenomes amplification of DNA from filter paper blood spot samplesPowerPoint Presentation: DNA template preparation for sequencing Protein-primed DNA amplification In situ genotyping with padlock probes Recombination based-cloning Cell-free cloning of lethal DNA RNA-primed DNA amplificationPowerPoint Presentation: Inactivation Inactivated by heating at 65°C for 10 min. Inhibition Inhibitors: aphidicolin , N 2 -(p-n- butylphenyl )- dGTP ( BuPdGTP ), 2-(p-n- butylanilino )- dATP ( BuAdATP ) (20). Molecular Weight 66.7 kDa monomer Quality Control The absence of endodeoxyribonucleases confirmed by appropriate quality tests. Source E.coli cells with a cloned gene 2 of Bacillus subtilisphage phi29.T4 DNA Polymerase: T4 DNA Polymerase T4 DNA Polymerase, a template-dependent DNA polymerase, catalyzes 5'-3' synthesis from primed single-stranded DNA. The enzyme has a 3'-5' exonuclease activity, but lacks 5'-3' exonuclease activity. Highlights Stronger 3'-5' exonuclease activity on single-stranded than on double-stranded DNA and greater (more than 200 times) than DNA polymerase I, E. coli, and Klenow fragment Active in restriction enzyme, PCR, RT and T4 DNA Ligase buffersPowerPoint Presentation: Applications Blunting of DNA ends: fill-in of 5'-overhangs or/and removal of 3'- overhangs Blunting of PCR products with 3'-dA overhangs Synthesis of labeled DNA probes by the replacement reaction Oligonucleotide-directed site-specific mutagenesis Ligation-independent cloning of PCR productsPowerPoint Presentation: Inactivation Inactivated by heating at 75°C for 10min. Inhibition Inhibitors: metal chelators, nucleotide analogs 2(p-n-butylanilino)-dATP, N2-(p-n-butylphenyl)-dGTP), SH-blocking compounds (see Reference 7) Molecular Weight 104kDa monomer Quality Control The absence of endodeoxyribonucleases confirmed by appropriate quality tests. Source E.coli cells with a cloned gene 43 of bacteriophage T4T7 DNA Polymerase: T7 DNA Polymerase T7 DNA Polymerase, a template dependent DNA polymerase, catalyzes DNA synthesis in the 5'=>3' direction. It is a highly processive DNA polymerase allowing continuous synthesis of long stretches of DNA. The enzyme also exhibits a high 3'=>5' exonuclease activity towards single- and double-stranded DNA . Assays at 37°C require only short incubation timesPowerPoint Presentation: Highlights Strong 3’=>5’ exonuclease activity , approximately 1000-fold greater than Klenow Fragment ( see Reference 1) Active in restriction enzyme buffersPowerPoint Presentation: Applications Purification of covalently closed circular DNA by removal of residual genomic DNA Primer extension reactions on long templates DNA 3'-end labeling Strand extensions in site-directed mutagenesis Fill-in blunting of 5'-overhang DNA Second strand synthesis of cDNA In situ detection of DNA fragmentation associated with apoptosisPowerPoint Presentation: Inactivation Inactivated by heating at 75°C for 10 min. Inhibition Inhibitors: metal chelators , modification reagents (acetic anhydride, N- ethylmaleimide inactivate the 3'=>5' exonuclease activity but not the polymerase activity) (see Reference 5) Molecular Weight The T7 DNA Polymerase is composed of two subunits: an 80 kDa polypeptide (the product of gene 5 of bacteriophage T7) and a 12 kDa thioredoxin (from the trxA gene of E. coli). Quality Control The absence of endodeoxyribonucleases is confirmed by the appropriate quality test. Source Two E. coli strains, one with the cloned gene 5 of bacteriophage T7, and the other with the cloned trxAgene of E. coli.Terminal Deoxynucleotidyl Transferase: Terminal Deoxynucleotidyl Transferase Terminal Deoxynucleotidyl Transferase ( TdT ), a template-independent DNA polymerase, catalyzes the repetitive addition of deoxyribonucleotides to the 3'-OH of oligodeoxyribonucleotides and single-stranded and double-stranded DNA . TdT requires an oligonucleotide of at least three nucleotides to serve as a primer. With RNA as template TdT shows variable performance which strongly depends upon the tertiary structure of acceptor RNA 3'-end and the nature of nucleotide. Generally, it is lower than using DNA as a template .PowerPoint Presentation: Due to the presence of CoCl 2, the TdT Reaction Buffer is incompatible with downstream applications. It is necessary to remove CoCl 2 from the reaction mixture by spin column or phenol/chloroform extraction and subsequent ethanol precipitationPowerPoint Presentation: Highlights Incorporates modified nucleotides (e.g., fluorescein-, biotin-, aminoallyl-labeled nucleotides) Applications Production of synthetic homo- and heteropolymers Homopolymeric tailing of linear duplex DNA with any type of 3'-OH terminus Oligodeoxyribonucleotide and DNA labeling 5 '-RACE (Rapid Amplification of cDNA Ends ) In situ localization of apoptosisPowerPoint Presentation: Inactivation Inactivated by heating at 70°C for 10 min or by addition of EDTA. Inhibition Inhibitors: metal chelators, ammonium, chloride, iodide, phosphate ions Quality Control The absence of endo -, exodeoxyribonucleases , phosphatases and ribonucleases confirmed by appropriate quality tests. Source E.coli cells carrying a cloned gene encoding calf thymus terminal deoxynucleotidyl transferase.Terminal Transferase (TdT): Terminal Transferase ( TdT ) Protruding, recessed or blunt ended double or single stranded DNA molecules serve as a substrate for TdT . TdT is isolated and purified from an E. coli strain carrying the cloned terminal transferase gene from calf thymus . Highlights Cloned and produced in E.coli Excellent stability and purity compared to native TdT EconomicalPowerPoint Presentation: Applications Addition of homopolymeric tails to plasmid DNA and to cDNA Double- or single-stranded DNA 3´-termini labeling with radioactively labeled or non-radioactively labeled nucleotides Addition of single nucleotides to the 3´ ends of DNA for in vitro mutagenesis Production of synthetic homo- and heteropolymers RACE (Rapid Amplification of cDNA Ends) In situ Localization of Apoptosis Resolving gel compressions and artifact banding in DNA sequencingDNA ligase – joining DNA molecules: DNA ligase – joining DNA molecules DNA ligase is an important cellular enzyme, as its function is to repair broken phosphodiester bonds that may occur at random or as a consequence of DNA replication or recombination. In genetic engineering it is used to seal discontinuities in the sugar—phosphate chains that arise when recombinant DNA is made by joining DNA molecules from different sources. It can therefore be thought of as molecular glue, which is used to stick pieces of DNA together.PowerPoint Presentation: This function is crucial to the success of many experiments, and DNA ligase is therefore a key enzyme in genetic engineering. The enzyme used most often in experiments is T4 DNA ligase, which is purified from E. coli cells infected with bacteriophage T4. Although the enzyme is most efficient when sealing gaps in fragments that are held together by cohesive ends, it will also join blunt-ended DNA molecules together under appropriate conditions.PowerPoint Presentation: The enzyme works best at 37◦ C, but is often used at much lower temperatures (4--15◦C) to prevent thermal denaturation of the short base-paired regions that hold the cohesive ends of DNA molecules together. The ability to cut, modify, and join DNA molecules gives the genetic engineer the freedom to create recombinant DNA molecules. The technology involved is a test-tube technology, with no requirement for a living system. However, once a recombinant DNA fragment has been generated in vitro , it usually has to be amplified so that enough material is available for subsequent manipulation and analysis.PowerPoint Presentation: Amplification usually requires a biological system, unless the polymerase chain reaction ( PCR ) is used. We must, therefore, examine the types of living systems that can be used for the propagation of recombinant DNA molecules. DNA ligase is essentially ‘molecular glue’; with restriction enzymes, it provides the tools for cutting and joining DNA molecules.Ligases: Ligases Fast and efficient ligation of DNA and RNA. T4 DNA Ligase T4 RNA LigaseT4 DNA Ligase: T4 DNA Ligase T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA. The enzyme repairs single-strand nicks in duplex DNA, RNA, or DNA/RNA hybrids. It also joins DNA fragments with either cohesive or blunt termini, but has no activity on single-stranded nucleic acids. The T4 DNA Ligase requires ATP as a cofactor .PowerPoint Presentation: Binding of T4 DNA Ligase to DNA may result in a band shift in agarose gels. To avoid this, incubate samples with 6X DNA Loading Dye & SDS Solution at 70°C for 5 min or 65°C for 10 minutes and chill on ice prior to electrophoresis. The volume of the ligation reaction mixture should not exceed 10% of the competent cell volume in the transformation process. Prior to electro-transformation, remove T4 DNA Ligase from the ligation mixture by spin column or chloroform extraction. The extracted DNA can be further precipitated with ethanol.PowerPoint Presentation: Highlights Active in restriction enzyme, PCR, and RT buffers (when supplemented with ATP) Fast – sticky-end ligation is completed in 10 minutes at room temperature Supplied with PEG solution for efficient blunt-end ligationPowerPoint Presentation: Applications Cloning of restriction enzyme generated DNA fragments Cloning of PCR products Joining of double-stranded oligonucleotide linkers or adaptors to DNA Site-directed mutagenesis Amplified fragment length polymorphism (AFLP) Ligase-mediated RNA detection Nick repair in duplex DNA, RNA or DNA/RNA hybrids Self-circularization of linear DNA.PowerPoint Presentation: Inhibition and Inactivation Inhibitors: T4 DNA Ligase is strongly inhibited by NaCl or KCl if the concentration exceeds 200 mM Inactivated by heating at 65°C for 10 minutes or 70°C for 5 minutes Molecular Weight 55.3 kDa monomer Quality Control The absence of endo-, exodeoxyribonucleases, phosphatases, and ribonucleases confirmed by appropriate quality tests. Functionally tested for the capacity to join cohesive- and blunt-end DNA fragments. Source E. coli cells with a cloned gene 30 of bacteriophage T4T4 RNA Ligase: T4 RNA Ligase T4 RNA Ligase catalyzes the ATP-dependent intra- and intermolecular formation of phosphodiester bonds between 5'-phosphate and 3'-hydroxyl termini of oligonucleotides, single-stranded RNA and DNA. The minimal substrate is a nucleoside 3',5'-biphosphate in intermolecular reaction and oligonucleotide of 8bases in intramolecular reaction. The recommended BSA concentration in the reaction mixture is 0.1mg/ mL.PowerPoint Presentation: Applications RNA 3'-end labeling with cytidine 3',5'- bis [alpha- 32 P ] phosphate Joining RNA to RNA Synthesis of oligoribonucleotides and oligodeoxyribonucleotides Specific modifications of tRNAs Oligodeoxyribonucleotide ligation to single-stranded cDNAs for 5' RACE (Rapid Amplification of cDNA Ends ) Site-specific generation of composite primers for PCRPowerPoint Presentation: Inactivation Inactivated by heating at 70°C for 10min. Inhibition Inhibitors: metal chelators, SH group-modifying reagents (8) Molecular Weight 43.6kDa monomer Quality Control The absence of ribonucleases , exodeoxyribonucleases , endodeoxyribonucleases , and phosphatases confirmed by appropriate quality tests. Source E.coli cells with a cloned gene 63 of bacteriophage T4PowerPoint Presentation: Other EnzymesRiboLock RNase Inhibitor: RiboLock RNase Inhibitor RiboLock RNase Inhibitor inhibits the activity of RNases A, B, and C by binding them in a noncompetitive mode at a 1:1 ratio. It does not inhibit eukaryotic RNases T1, T2, U1, U2, CL3 as well as prokaryotic RNases I and H. Highlights Performs under a wide range of reaction conditions Protects RNA from degradation at temperatures up to 55° CPowerPoint Presentation: Inactivation Inactivated by heating at 75°C for 10 min. Residual activity detectable after 10 min heating at 70°C. Inhibition Inhibitors: common denaturants (SDS, urea and all oxidizing reagents (p- chloromercuribenzoate , dissolved oxygen, ions in their higher oxidation states) strongly inhibit RiboLock RNase Inhibitor and release the RNase bound. Molecular Weight 49.6 kDa monomer Quality Control The absence of ribonucleases, endo-, exodeoxyribonucleases and phosphatases confirmed by appropriate quality tests. Functionally tested in RNA and cDNA synthesis. Source E.coli cells with a cloned gene encoding mammalian ribonuclease inhibitor.Phosphatases & Kinases: Phosphatases & Kinases Alkaline phosphatase and polynucleotide kinase for DNA and RNA dephosphorylation or phosphorylation . FastAP Alkaline Phosphatase T4 Polynucleotide KinaseT4 Polynucleotide Kinase: T4 Polynucleotide Kinase Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the gamma-phosphate from ATP to the 5'-OH group of single- and double-stranded DNAs and RNAs, oligonucleotides or nucleoside 3'-monophosphates (forward reaction ). The reaction is reversible. In the presence of ADP T4 Polynucleotide Kinase exhibits 5'-phosphatase activity and catalyzes the exchange of phosphate groups between 5'-P-oligo-polynucleotides and ATP (exchange reaction ).PowerPoint Presentation: The enzyme is also a 3'-phosphatase Polyethylene glycol (PEG) and spermidine improve the rate and efficiency of the phosphorylation reaction . PEG is used in the exchange reaction mixture. As T4 Polynucleotide Kinase is inhibited by ammonium ions, use sodium acetate to precipitate DNA prior to phosphorylationPowerPoint Presentation: Highlights Active in restriction enzyme, RT, and T4 DNA Ligase buffers Applications Labeling of nucleic acids' 5'- termini to be used as: probes for hybridization probes for transcript mapping markers for gel electrophoresis primers for DNA sequencing primers for PCRPowerPoint Presentation: 5'-phosphorylation of oligonucleotide, PCR products, other DNA or RNA prior to ligation Phosphorylation of PCR primers Detection of DNA modification by the [ 32 P]- postlabeling assay Removal of 3'-phosphate groupsPowerPoint Presentation: Inactivation Inactivated by heating at 75°C for 10min or by addition of EDTA. Inhibition Inhibitors: metal chelators, phosphate and ammonium ions, KCl and NaCl at a concentration higher than 50mM Molecular Weight The enzyme is a homotetramer. It consists of four identical subunits of 28.9kDa. Quality Control The absence of endo -, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested for labeling 5'-termini of DNA.conclusion: conclusion These are the modifying enzymes represent the cutting and joining functions in DNA manipulation and genetic engineering.references: references You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.