An enzyme, essential for dna replication, that catalyzes the covalent bonding of adjacent dna strands. Kinetics and thermodynamics of nick sealing by t4 dna ligase. It also joins dna fragments with either cohesive or blunt term. The 10x buffer supplied with this enzyme has a composition of 250mm trisacetate ph 7. Efficient dna nick sealing catalyzed by t4 dna ligase was carried out on a modified dna template in which an intercalator such as azobenzene had been introduced.
Dna unwinding and inhibition of t4 dna ligase by anthracyclines article pdf available in nucleic acids research 169. A small amount of dna mixed with a heatresistant dna polymerase, dna nucleotides, and a few other ingredients replicates repeatedly in test tube. Dna ligases are a useful tool in generating recombinant dna sequences. Enzyme product 5 is identical to the starting substrate. Below this are 4 enzymatic products made from the starting substrate enzyme products 14. The cohesive end unit is equivalent to the nickclosing unit of barany et al. It plays a role in repairing singlestrand breaks in duplex dna in living organisms, but some forms such as dna ligase iv may specifically repair doublestrand breaks i. The minimal substrate is a nucleoside 3,5biphosphate in intermolecular reaction and oligonucleotide. The enzyme repairs singlestrand nicks in duplex dna, rna, or dnarna hybrids.
Most ligases employ atp as a cofactor and form covalent adenylyl intermediate not drawn for simplicity, which activates the 5. T4 dna ligase catalyzes the formation of phosphodiester bonds in the presence of atp between doublestranded dnas with 3hydroxyl and 5phosphate termini. Binding of t4 dna ligase to dna may result in a band shift in agarose gels. T4 dna ligase catalyzes the joining of two strands of dna between the 5. Jun 01, 1997 atpdependent dna ligases are essential enzymes in both dna replication and dna repair processes. Despite extensive purification of t4 dna ligase, attempts to crystallize the protein, both with and without cofactor, have been unsuccessful. Different substrate specificities of the two dna ligases. Structural biochemistryt4 dna ligase wikibooks, open books. Nick sealing by t4 dna ligase on a modified dna template. Hydrogen bonds between base pairs of antiparallel strands are broken. Unlike t4 rna ligase 1, t4 rna ligase 2 is much more active joining nicks on double stranded rna than on joining the ends of single stranded rna. One weiss unit is approximately equivalent to 22 units.
Dna and detected by western blot or mass spectrometry. Thermo scientific t4 dna ligase product help molecular cloning. T4 dna ligase catalyzes the formation of a phosphodiester bond between the terminal 5 phosphate and a 3 hydroxyl groups of duplex dna or rna. T4 dna ligase the enzyme efficiently joins blunt and. Gently mix the reaction by pipetting up and down and microfuge briefly. Thermo scientific products help scientists around the world meet the challenges they face every day. Thermo scientific t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna.
T3 dna ligase is an atpdependent ds dna ligase from bacteriophage t3. Put the following steps of dna replication in chronological order. Ligation protocol with t4 dna ligase m0202 protocols. Measure amount of dna in sample using nanodrop spectrophotometer. Thermo scientific t4 rna ligase catalyzes the atpdependent intra and intermolecular formation of phosphodiester bonds between 5phosphate and 3hydroxyl termini of oligonucleotides, singlestranded rna and dna. Greetings i have been struggling to ligate a 555 bp insert into a 6. Heat inactivate at 65c for 10 min or at 70c for 5 min. T4 dna ligase catalyzes the formation of phosphodiester bonds between neighbouring 3hydroxyl and 5phosphate ends in doublestranded dna. One t4 dna ligase cohesive end unit is equivalent to approximately 3 cohesive end units as measured with a lambdahind iii dna fragment substrate in 1x t4 dna ligase reaction buffer. Therefore, invitrogen recommends the enzyme be kept at 20 c until within 510 minutes of use and returned immediately to 20 c after use.
Kinetics and thermodynamics of nick sealing by t4 dna ligase article pdf available in european journal of biochemistry 27021. Thermo scientific t4 dna ligase product help molecular. Therefore, invitrogen recommends the enzyme be kept at 20c until within 510 minutes of use and. Cohesive ends, blunt ends, and nick sealing can all be efficiently catalyzed by t3 dna ligase 1. The enzyme efficiently joins blunt and cohesive ends and repairs singlestranded nicks in duplex dna, rna or dnarna hybrids. Note t4 dna ligase is active in pcr and restriction. About 3 years ago several companies introduced so called rapid ligase.
The thermo scientific 10x t4 dna ligase buffer is used with t4 dna ligase. For example, dna fragments are cut with restriction enzymes and then recombined with dna ligase. T4 dna ligase will seal nicks for these dna substrates. One nterminal and two cterminal deletion mutants were expressed in escherichia coli as histidine tagged proteins. The enzyme repairs singlestrand nicks in duplex dna, rna or dnarna hybrids. Different substrate specificities of the two dna ligases of mammalian cells. The t7 dna ligase reaction buffer should be thawed and resuspended at room temperature. Pdf dna unwinding and inhibition of t4 dna ligase by. Dna ligase is a specific type of enzyme, a ligase, ec 6. Y90001 5x t4 dna ligase buffer buffer composition 5x concentration1. These enzymes show different molecular weights and heat labilities, and antibodies against ligase i do not inhibit ligase ii. An additional mutant bore a substitution of lys159 in the active. T4 dna ligase the enzyme efficiently joins blunt and cohesive. In my lab we use rapid ligases from roche and fermentas.
This higher concentration is required for rapid ligation of blunt ends. Zymo purify restriction digested dna see zymo procedure. It will catalyze the formation of a phosphodiester bond between adjacent 5. From routine analysis to pioneering discoveries, our innovations help scientists solve complex analytical challenges, empowering them to conduct the work they need to do, the way they want to do it. Rev 052002 this product is distributed for laboratory research only. Dna ligase iv it catalyzes the final step in the nonhomologous end joining dna doublestrand break repair pathway. At a 1x concentration this reaction buffer assures optimal activity of the enzyme. Catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex dna or rna. A similar structure, that of t7 dna ligase, has been solved subramanya et al. Chapter 2 structures of nucleic acids nucleic acids. Less than 1% of white colonies were detected after transformation of competent. T4 dna ligase can be used to join dna fragments with staggered or blunt ends. D2886 catalyzes the formation of a phosphodiester bond between the terminal 5.
T4 dna ligase catalyzes the formation of a phosphodiester bond between the terminal 5. No conversion of covalently closed circular dna to product. T4 dna ligase recombinant form of the enzyme from t4 phage. Dna ligase iv is the nhejspecific ligase grawunder et al. For blunt ends, use 1 l of t4 dna ligase in a 20 l reaction for 2 hours or 1 l high concentration t4 dna ligase for 10 minutes. This enzyme can be purchased from many different companies, usually from the same companies that produce restriction enzymes. Atpdependent dna ligases are essential enzymes in both dna replication and dna repair processes. Here we report a functional characterization of the t4 dna ligase. Singlestranded nucleic acids are not substrates for this enzyme. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dna rna hybrids 1.
Singlestranded binding proteins attach to dna strands. Singlestranded nicks in doublestranded dna are also closed. The figure at the top represents a double stranded dna molecule that has a single missing phosphodiester bond a nick. The enzyme efficiently joins blunt and cohesive ends and repairs single stranded nicks in duplex dna, rna or dnarna hybrids 1. Alternatively, the protein may be labeled with an affinity tag or the dna. In this case, the dna interacting with the target protein is detected by southern blot or through pcr analysis. Size units m421a 100 m421f 500 enzyme storage buffer. The t4 dna ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5phosphate and 3hydroxyl termini in duplex dna or rna with blunt or cohesiveend termini. Structural biochemistryt4 dna ligase wikibooks, open. The enzyme repairs singlestrand nicks in duplex dna, rna, or dna rna hybrids. Set up the following reaction in a microcentrifuge tube on ice. The enzyme has also been shown to catalyze the joining of rna to either a dna or rna strand in a duplex molecule, but will not join singlestranded.
T4 dna polymerase to create blunt ends keep enzyme on ice if we use frequently, we need to aliquot it since it is sensitive to multiple freeze thaw cycles 1. The 10x buffer has been tested for absence of endo, exodeoxyribonucleases, ribonucleases and functionally tested in dna ligation. Learn more about how this product is being used in the product citation tool. T4 dna ligase is an enzyme that is encoded by the bacteriophage known as t4.
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