DNA ligation

DNA Ligation Kit Ver.2 (BioDynamics Laboratory Inc., Bunkyo-ku, Tokyo, Japan). 

Three microliters of this solution and 1 pL of the vector solution are mixed in a 96-well plate. Four microliters of 2x Ligation Buffer and 0.4 pL of Ligase Mixture from a DNA Ligation Kit Ver.2 is blended and added to the DNA mixture. After pipetting twice using an 8-channel autopipetter, this ligation reaction mixture set is incubated at 16°C for 30 min. 

After linearization of the transfer vector with alkaline phosphatase, the target gene DNA is ligated into the linearized transfer vector by using DNA Ligation Kit Ver 2.1 (TAKARA BIO INC., Japan) (tee Note 5). 

Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, Ostman A, Landegren U, Protein detection using proximity-dependent DNA ligation assays, Nat. Biotechnol., 20 413-411, 2002. 

Day 1 Restriction Endonuclease Digestion of pUC19 and pUC4K, Ethanol Precipitation of DNA Fragments, and DNA Ligation... 

Add 1 ml of ice cold 70% ethanol to the DNA pellet, centrifuge at 4°C for 5 min, and carefully remove the supernatant from the precipitated DNA pellet. This step is done to remove the salt that was added to the DNA to aid in the precipitation. This salt, if not removed, could interfere with the DNA ligation reaction. 

Single-strand breaks are generated by radiation. However, these breaks cannot be repaired by a simple DNA ligation, which requires a 3 OH group and a 5 phosphate group at the nick. Studies indicate that, at these breaks, phosphate groups are... 

Figure 27.29. DNA Ligase Mechanism. DNA ligation proceeds by the transfer of an AMP unit first to a lysine side chain on DNA ligase and then to the 5 -phosphate group of the substrate. The AMP unit is released on formation of the phosphodiester linkage in DNA.

Keywords QCM Enzyme reaction DNA polymerase DNA hydrolysis DNA ligation DNA cleavage Glycosylation Phosphorylation Mutation of enzyme... 

DNA ligase has been used as a tool for in vitro DNA manipiflation and cloning techniques. The reaction mechanism of DNA ligation has been mainly studied by measuring the combination of Rl-labeled substrates and gel electrophoresis. Despite several improvements, these techniques have still some difficulties such as the requirement of isotope-labehng of probes and of special techniques. 

Figure 8.11 Platelet-derived growth factor B (PDGF-BB) detection using a proximity-dependent DNA ligation assay. [From Fredriksson et al. (2002).]...

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