Recombinant DNA, Part B by Ray Wu, Lawrence Grossman, Kivie Moldave

By Ray Wu, Lawrence Grossman, Kivie Moldave

FROM THE PREFACE: fascinating new advancements in recombinant DNA study let the isolation and amplification of particular genes or DNA segments from virtually any residing organism. those new advancements have revolutionized our ways to fixing complicated organic difficulties and feature spread out new chances for generating new and higher items within the components of overall healthiness, agriculture, and industry.Volumes a hundred and one zero one complement Volumes sixty five and sixty eight of tools in Enzymology. over the last 3 years, many new or superior equipment on recombinant DNA or nucleic acids have seemed, and they're integrated in those volumes. quantity a hundred covers using enzymes in recombinant DNA learn, enzymes affecting the gross morphology of DNA, proteins with really good features performing at particular loci, new tools for DNA isolation, hybridization, and cloning, analytical tools for gene items, and mutagenesis: in vitro and in vivo. quantity one zero one contains sections on new vectors for cloning genes, cloning of genes into yeast cells, and platforms for tracking cloned gene expression.

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H. Huang, C. M. Farnet, K. C. Ehrlich, and M. Ehlich, Nucleic Acids Res. 10, 1579 (1982). u H. Youssoufian and C. Mulder, J. Mol. Biol. 150, 133 (1981). 16 ENZYMES IN RECOMBINANT D N A [1] When 30 type II restriction endonucleases were separately incubated with Xanthomonas oryzae phage XP12 DNA, all cytosine residues of which are modified to 5-methylcytosine, only TaqI cleaved efficiently. When bacteriophage T4 DNA, which contains only 5-hydroxymethylcytosine, but not cytosine, was tested, again only TaqI cleaved, although inefficiently.

22 Methylation of nucleotides within restriction endonuclease recognition sequences, occurring almost exclusively as 5-methylcytosine or N6-methyladenine, prevented most T A B L E IV METHYLATED D N A s AS SUBSTRATES FOR RESTRICTION ENDONUCLEASES a S e q u e n c e s containing 5-methylcytosine or N6-methyladenine o Enzyme Aos lI Ava I Ava I I Cleaved Not cleaved References -- GPumCGPyC d, e -- CPymCGPuG f -- GG(A)CmC g (T) BstNI CmC(A)GG -- h (T) EcoRII -- CmC(A)GG h, i (T) Hae I I HaelII HaplI Hha I HpalI MspI Pst I Pvu I I SalI SrnaI Taql Xhol Xma I Barn HI Bglll DpnI Dpn I I EcoRI Fnu E1 HindlI HindlII HpaI MboI MbolI Sau3AI TaqI -- PuGmCGCPy e, f GGCmC GGmCC j, k -- CmCGG e, l -- GmCGC f, j mCCGG C~"CGG j, l CmCGG mCCGG -- mCTGCAG I, m n -- mCAGCTG n -- GT~CGAC d, e -- CCmCGGG TmCGA -- e, u -- CTmCGAG CCmCGGG -- u GGmATCC -- g, p AGmATCT -- p GmATC c -- o d, e o, q -- GmATC q r -- GAmATTC GmATC -- s -- GTPyPumAC k -- mAAGCTT k -- GTTAmAC -- GmATC t p, s -- GAAGmA GmATC -- o, p g -- TCGmA d, o [1] USE OF TYPE II RESTRICTION ENDONUCLEASES 15 enzymes from cleaving.

DNA associations, such as the "sticky ends" of lambda DNA. When the products of the reaction are to be used subsequently for kinasing, ligation, or sequencing, the reaction can be terminated, in some cases, by heat inactivation of the enzyme, or more reliably by phenol extraction of the DNA fragments. Some enzymes such as E c o R I 9 or HaeII 8 are irreversibly inactivated by exposure to 65 ° for 5 min, whereas Tth139 and HindII148 remain active after this treatment. 1 M Tris-HCl (pH 8). 5 M ammonium acetate and two volumes of ethanol for 30 min at - 7 0 °.

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