Analysis and optimization of recombinant DNA joining reactions.
The statistical segment length of duplex DNA was determined in phage T4 ligase (poly(deoxyribonucleotide): poly(deoxyribonucleotide) ligase (AMP forming), EC 6.5.1.1) buffer (50 mM-Tris . HCl (pH 7.8), 20 mM-dithiothreitol, 10 mM-MgCl2, 1 mM-ATP) at 12 degrees C to be 1030(+/- 116) A. This result was obtained by electron microscopic examination of the molecular distributions generated by T4 ligase-mediated joining of EcoRI-cleaved pBR322 DNA. This value of the statistical segment length was utilized in an extension of the Jacobson-Stockmayer theory on the probability of intramolecular cyclization in order to optimize DNA joining reactions that are of great utility in recombinant DNA experiments. Five cloning systems were analyzed: circular plasmid vectors that had been linearized with one or two restriction endonucleases, circular plasmids that had been tailed with deoxyhomopolymers before joining, lambda-type cloning vectors and cosmids. The results are tabulated for convenient use in molecular cloning experiments.
Legerski
R J
RJ
Robberson
D L
DL
D003001:Cloning, Molecular; D011088:DNA Ligases; D004262:DNA Restriction Enzymes; D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral; D004587:Electrophoresis, Agar Gel; D008433:Mathematics; D008722:Methods; D008854:Microscopy, Electron; D010957:Plasmids; D011120:Polyomavirus; D013539:Simian virus 40; D018139:Simplexvirus
D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral; D004262:DNA Restriction Enzymes; D011088:DNA Ligases