Recombinant DNA Part A by Kaplan N.P. (ed.), Colowick N.P. (ed.), Wu R. (ed.)

By Kaplan N.P. (ed.), Colowick N.P. (ed.), Wu R. (ed.)

The looks of one other quantity in that very good sequence, equipment in Enzymology, is often a reason for appreciation in the event you desire to effectively perform a specific process or arrange an enzyme or metabolic intermediate with out the tiresome prospect of looking through unusual literature and maybe making a choice on an unproven procedure which isn't simply reproduced.

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This reaction, called blunt-end joining, is a powerful method for structuring DNA molecules in vitro (see below) but proceeds much less readily than nick sealing. Blunt-end joining is not linearly dependent on enzyme concentration but increases greatly at higher enzyme levels. Although T4 RNA ligase does not catalyze blunt-end joining, it markedly stimulates the reaction, particularly at low DNA ligase concentrations. 41 As much as a 20-fold increase in the rate was observed at low DNA ligase concentrations, and the stimulation was specific for blunt-end joining because RNA ligase had only a marginal influence on the joining of cohesive ends.

Biol. Chem. 244, 594 (1969). ~2W. R6wekampand R. Firtel, unpublishedresults. 48 ENZYMES IN RECOMBINANT DNA RESEARCH [3] Pstl CTGCAG Vector G A C G T C Vector T PstI Pstl Cleavage G 3 ' A C G T C Vector Vector C T G C A 3 ' G I Poly (dG) addition G Vector 3'GGGGGGGGACGTC Vector C T G C A G G G G G G G G 3 ' G + Insert C C C C C C C C 3 ' 3'CCCCCCCC - - ~Annealing Vector CTGCAGGGGGGGG ~ G 3'CCCCCCCC - - CCCCCCCCY G Vector GGGGGGGGACGTC Cloning PstI I Pstl CTGCAGGGGGGGG ~ CCCCCCCCTGCAG Vector msert GGGGGGGGACGTC Vector G A C G T C C C C C C C C Psd Pstl Hybrid DNA SCHEME 1 phate and to the presence of other contaminating nucleotide species.

The latter methods are easily reversible, and Ohtsuka et al. s6 used this approach to synthesize a heptadecanucleotide corresponding to bases 61-77 of E. coli tRNA fiet. The smallest monoaddition donors are the mononucleoside 3',5'-bisphosphates pCp, pAp, pUp, pGp, pdCp, pdAp, pdTp, pdUp, and pdGp. They add to the 3'-terminus of a number of different acceptors including tRNA, mRNA, and double- and singlestranded viral R N A Y ,ss These studies are examples of the important application of RNA ligase technology to the precise engineering of naturally derived RNA molecules.

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