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DNA Ligation
DNA ligation is the joining of two nucleic acid fragments through the action of an enzyme. Several factors can affect the ligation reaction, including the concentration of enzyme, DNA, and cofactors like ATP or NAD+. The DNA concentration is particularly important, as higher concentrations favor intermolecular ligation between separate DNA molecules, while lower concentrations favor intramolecular ligation where a DNA molecule joins its own ends. DNA ligase carries out ligation through a three-step catalytic mechanism involving adenylation of the enzyme and two phosphoryl transfers.
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DNA Ligation
- 1. 1 DNA Ligation • Ligationis the joining of two nucleic acid fragments through the action of an enzyme . • The ends of the fragments are joined together by the formation of phosphor-di-ester bonds between the 3’- hydroxyl of one DNA terminus with the 5’-phosphoryl of another . • A co-factor is generally involved in the reaction, and this usually ATP or NAD+
- 2. 2 Factor Affecting Ligation •Factor that affecting an Enzyme-mediated chemical reaction would naturally affect a ligation reaction . Such as :- ◘ The concentration of enzyme and the reactants . ◘ The temperature of the reaction . ◘ The length of time of incubation . ○◘○ ♦ Ligation is complicated by the fact that the desired ligation products for most ligation reaction should be between two different DNA molecules . ♦ And the reaction involves both inter- and intra-molecular reactions . ♦ And that an additional annealing step is necessary for efficient ligation .
- 3. 3 DNA Concentration • Theconcentration of DNA can affect the rate of ligation . • And whether the ligation is an inter-molecular or intra- molecular reaction . • Ligation involves joining up the ends of a DNA with other ends , however , each DNA fragment has two ends . • And If the ends are compatible ) (متكاملين , a DNA molecule can circularize by joining its own ends .
- 4. 4 • At HighDNA concentration , there is a greater chance of forming inter-molecular ligation . • While at lower DNA concentration , intra-molecular reaction that circularizes the DNA is more likely . • The Transformation efficiency of linear DNA is much lower than circular DNA , and for the DNA to circularize , the DNA concentration should not be too high . • As a general rule , the total DNA concentration should be less than 10 µg/ml .
- 5. 5 • the relativeconcentration of the DNA fragments , as well as their length , are also factors that can affect whether intermolecular or intermolecular reactions are favored . • The concentration of DNA can be artificially increased by adding condensing agents such as cobalt Hexamine and biogenic polyamines such as spermidine. • or by using crowding agents such as polyethelene glycol ( PEG ) which also increase the effective concentration of enzymes .
- 6. 6 Ligase concentration ♣ Thehigher the ligase concentration , the faster the rate of ligation . ♣ blunt-ends ligation is much less afficient than sticky end ligation . ♣ So a Higher concentration of ligase is used in blunt-end ligation . ♣ High DNA ligase concentration my used in conjunction with PEG for faster ligation
- 7. 7 Tempertaure ♥ Two issuesare involved when considering the temperture of a ligation reaction . ♦ First : the optimum temperture for DNA ligase activities which is 37o C . ♦ Second : Tthe melting temperture ( TM ) of the DNA ends to be ligated . • The melting temperture is dependent on the length and base composition of the DNA overhang . • The greater the number of G and C , The higher the TM since there are three hydrogen bonds formed , between G-C base pair compared to two For A-T base . • Ligation reaction to proceed the ends should be stably annealed . • and in ligation experiments , the TM of the DNA ends is generally much lower than 37o C . • The Optimal temperature for ligation cohesive ends is therefore a compromise between) بينهم ما وسط ( the best temperature For DNA ligase activity and TM where the ends can associate . • Most protocols simply choose 12-16 o C , room temperature , or 4o C .
- 8. 8 Buffer composition • TheIonic strength of the buffer used can affect the ligation . • The kinds of cations presence can also influence the ligation reaction . • For example , excess amount of NA+ can cause the DNA to become more rigid ) (صلب and increase the likelihood )(احتمالية of intermolecular ligation . • At high concentration of Monovalent ()التكافؤ احادي cations ( >200 mM) Ligation can be almost completely inhibited. • The standard buffer used for ligation is designed to be minimize ionic effects .
- 9. 9 Mechanism of DNALigase • Sealing) ربط ( a nick ) فتحة او (شق in DNA might appear a simple task , but the reaction is exceedingly complex ) جدا (معقد . • require three distinct ) (واضح catalytic steps and two covalent intermediates . • The first step :- - enzyme adenylation is accomplished using either NAD+ ( in eubacteria ) or ATP , but both result in an AMP-linkage to the enzyme . • In The Second step :- - the AMP moiety is transferred to the 5’ terminus for attack the 3’ OH in the third and final phosphoryl transfer step , thereby sealing the nick .
- 10. 10 • To formthis stable reaction intermediate used a synthetic nicked duplex terminated with a 3’ dideoxynucleotide . • Thereby ) بذلك ( removing the critical 3’ OH group necessary to form the phosphodiester bond .
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