Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Cloning PCR Products: TOPO TA Cloning

(Protocol summary only for purposes of this preview site)

The topoisomerase of vaccinia virus, like other type IB topisomerases, relaxes DNA supercoils by transiently breaking and rejoining one strand of the DNA duplex. Nicking of the DNA duplex occurs as a result of nucleophilic attack on the DNA phophodiester bond by the OH group of tyrosine 274 of the topoisomerase. Cleavage of the target DNA strand occurs with high specificity at a pentapyrimidyl tract 5(C/T)CCTT, creating a stable 3-phosphotyrosyl-DNA intermediate (Shuman et al. 1989). Because bond energy is conserved, the reaction is readily reversible and the phosphotyrosyl-DNA intermediate can either (i) catalyze reformation of the original duplex or (ii) create a recombinant DNA by forming a phosphodiester bond with an acceptor DNA with a 5-OH tail complementary to that of the donor complex (see Fig. 1).


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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