Preface Recombinant DNA methods are powerful, revolutionary techniques for at least two reasons. First, they allow the isolation of single genes in large amounts from a pool of thousands or millions of genes. Second, the isolated genes or their regulatory regions can be modified at will and re- introduced into cells for expression at the RNA or protein levels. These attributes allow us to solve complex biological problems and to produce new and better products in the areas of health, agriculture, and industry. Volumes 153, 154, and 155 supplement Volumes 68, 100, and 101 of Methods in Enzymology. During the past few years, many new or im- proved recombinant DNA methods have appeared, and a number of them are included in these three new volumes. Volume 153 covers methods related to new vectors for cloning DNA and for expression of cloned genes. Volume 154 includes methods for cloning cDNA, identification of cloned genes and mapping of genes, chemical synthesis and analysis of oligodeoxynucleotides, site-specific mutagenesis, and protein engineer- ing. Volume 155 includes the description of several useful new restriction enzymes, detail of rapid methods for DNA sequence analysis, and a num- ber of other useful methods. RAY WU xiii Contributors to Volume 155 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. ASAD AHMED (14), Department of Genetics, University of Alberta, Edmonton, Al- berta, Canada T6G 2E9 AMY ARROW (15), Biotix Inc., Commerce Park, Danbury, Connecticut 06810 A. T. BANKIER (7), MedicalResearch Coun- cil Laboratory of Molecular Biology, Cambridge CB2 2QH, England B. G. BARKELL (7), Medical Research Council Laboratory of Molecular Biol- ogy, Cambridge CB2 2QH, England KIRK BAUMEISTER (11), E. I. du Pont de Nemours & Company, Inc., Central Re- search and Development Department, Experimental Station, Wilmington, Dela- ware 19898 STEPHEN BECK (18), Medical Research Council Laboratory of Molecular Biol- ogy, Cambridge CB2 2QH, England JUDITH BERMAN (32), Department of Bot- any, University of Minnesota, Twin Cit- ies, St. Paul, Minnesota 55108 HERMANr~ BUJARD (26), Zentrum far Mole- kularbiologie, Universitiit Heidelberg, D-6900 Heidelberg, Federal Republic of Germany CHARLES R. CANTOR (28), Departments of Genetics and Development, College of Physicians and Surgeons of Columbia University, New York, New York 10032 GEORGES F. CARLE (29), Department of Ge- netics, Washington University School of Medicine, St. Louis, Missouri 63110 MAIR E. A. CHURCHILL (33), Department of Chemistry, The Johns Hopkins Univer- sity, Baltimore, Maryland 21218 RICCARDO CORTESE (9), Uniuersitd di Na- poli, lstituto di Scienze Biochimiche, H Facoltd di Medicina, 80131 Napoli, Italy, and European Molecular Biology Labora- ix tory, Meyerhofstrasse 1, 6900 Heidelberg, Federal Republic of Germany PAMELA F. CRAIN (23), Department of Me- dicinal Chemistry, College of Pharmacy, The University of Utah, Salt Lake City, Utah 84112 RODERIC M. K. DALE (15), Biotix Inc., Commerce Park, Danbury, Connecticut 06810 LUCIANA DENTE (9), Universitd di Napoli, Istituto di Scienze Biochimiche, H Fa- coltd di Medicina, 80131 Napoli, Italy BERNHARD DOBBERSTEIN (26), European Molecular Biology Laboratory, D-6900 Heidelberg, Federal Republic of Ger- many BETH A. DOMBROSKI (33), Department of Chemistry, The Johns Hopkins Univer- sity, Baltimore, Maryland 21218 SHLOMO EISENBERG (32), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 FRED A. FAEOONA (21), Molecular Biology Department, Xytronyx, Inc., 6555 Nancy Ridge Drive, San Diego, California 92121 REINER GENTZ (26), Central Research Units, Hoffman-La Roche and Company AG, CH-4002 Basel, Switzerland R. S. GOODY (13), Department of Biophys- ics, Max-Planck Institut fiir medizinische Forschung, 6900 Heidelberg, Federal Re- public of Germany MARIE-THERESE HAEUPTLE (26), European Molecular Biology Laboratory, D-6900 Heidelberg, Federal Republic of Ger- many NAOHIRO HANYU (24), National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan X CONTRIBUTORS TO VOLUME 155 STEVEN HENIKOFF (12), Fred Hutchinson Cancer Research Center, Seattle, Wash- ington 98104 PHiLiP HIETER (22), Department of Molecu- lar Biology and Genetics, The John Hopkins University School of Medicine, Baltimore, Maryland 21205 DAVID E. HILL (34), Genetics Institute Inc., Cambridge, Massachusetts 02140 GERD HOBOM (1), Institute of Microbiology and Molecular Biology, Justus-Liebig University, D-6300 Giessen, Federal Re- public of Germany Guo-FAN HUNG (8), Shanghai Institute of Biochemistry, Academia Sinica, Shang- hai 200031, China LEROY E. HOOD (19), Division of Biology, California Institute of Technology, Pasa- dena, California 91125 HANS-DIETER HUNGER (20), Abteilung Mo- lekulare Humangenetik, Zentralinstitut fiir Molekularbiologie, Akademie der Wis- senschaften der DDR, 1115 Berlin-Buch, German Democratic Republic IBRAHIM IBRAHIMI (26), European Molecu- lar Biology Laboratory, D-6900 Heidel- berg, Federal Republic of Germany GABOR L. IGLOI (27), Institut fiir Biologie III der Universitdt Freiburg, D-7800 Frei- burg, Federal Republic of Germany RUDOLF JUNG (20), Zentralinstitut far Genetik und Kulturpflanzenforschung, Akademie der Wissenschaften der DDR, 4325 Gatersleben, German Democratic Republic ROBERT J. KAISER (19), Division of Biology, California Institute of Technology, Pasa- dena, California 91125 LAURANCE KAM (33), Department of Chem- istry, The Johns Hopkins University, Bal- timore, Maryland 21218 DOUGLAS KOSHLAND (22), Department of Embryology, Carnegie Institution of WashMgton, Baltimore, Maryland 21210 HANS KOSSEL (27), Institut far Biologie III der Universitdt Freiburg, D-7800 Frei- burg, Federal Republic of Germany P. A. KRIEG (25), Department of Zoology, University of Texas at Austin, Austin, Texas 78712 MANFRED KR6GER (1), Institute of Microbi- ology and Molecular Biology, Justus- Liebig University, D-6300 Giessen, Fed- eral Republic of Germany YOSHIYUKI KUCHINO (24), Biology Divi- sion, National Cancer Center Research Institute, TsukUi 5-1-1, Chuo-ku, Tokyo 104, Japan S. LABEIT (13), National Cancer Research Institute, Department of Cell Biology, Im Neuerheimerfeld, 6900 Heidelberg, Fed- eral Republic of Germany MICHAEL LANZER (26), Zentrum fiir Mole- kularbiologie, Universitdt Heidelberg, D-6900 Heidelberg, Federal Republic of Germany H. LEHRACH (13), Imperial Cancer Re- search Fund, Lincoln's Inn Fields, Lon- don WC2A 3PX, England LEONARD S. LERMAN (30, 31), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02138 F. I. LEWITTER (36), Life Sciences Re- search Systems, BBN Laboratories Incor- porated, 10 Moulton Street, Cambridge, Massachusetts 02238 TOM MANIATIS (31), Department of Bio- chemistry and Molecular Biology, Har- vard University, Cambridge, Massachu- setts 02138 MICHAEL MCCLELLAND (4, 5), Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 D. A. MELTON (25), Department of Bio- chemistry and Molecular Biology, Har- vard University, Cambridge, Massachu- setts 02138 TAPAN K. MISRA (10), Department of Mi- crobiology and Immunology, University of lllinois College of Medicine, Chicago, Illinois 60612 MICHAEL MUELLER (26), Zentrumfiir Mole- kularbiologie, Universitdit Heidelberg, CONTRIBUTORS TO VOLUME 155 xi D-6900 Heidelberg, Federal Republic of Germany KARY B. MULLIS (21), Molecular Biology Department, Xytronyx, Inc., 6555 Nancy Ridge Drive, San Diego, California 92121 RICHARD M. MYERS (31), Department of Physiology, School of Medicine, Univer- sity of California at San Francisco, San Francisco, California 94143 MICHAEL NELSON (5, 6), New England Biolabs, Inc., 32 Tozer Road, Beverly, Massachusetts 01915 SUSUMU NISHIMUaA (23, 24), National Cancer Center Research Institute, Tsuk(]i 5-1-1, Chuo-ku, Tokyo 104, Japan C. DAVID O'CONNOR (2), Department of Biochemistry, University of Southamp- ton, Southampton S09 3TU, England ARNOLD R. OLIPHANT (34, 35), Department of Biological Chemistry, Harvard Medi- cal School, Boston, Massachusetts 02115 MAYNARD V. OLSON (29), Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110 ZE-GUO PENG (16), National Cancer Insti- tute, Frederick Cancer Research Facility ~ Frederick, Maryland 21701 FRITZ M. POHL (18), Fakultiitfiir Biologie, Universitiit Konstanz, D-7750 Konstanz, Federal Republic of Germany Bo-QIN QIANG (3), Department of Biochem- istry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Acad- emy of Medical Sciences, Beijing, China W. P. RINDONE (36), Life Sciences Re- search Systems, BBN Laboratories Incor- porated, 10 Moulton Street, Cambridge, Massachusetts 02238 ANDRI~ ROSENTHAL (20), Abteilung Moleku- lare Humangenetik, Zentralinstitut far Molekularbiologie, Akademie der Wis- senschaften der DDR, 1115 Berlin-Buch, German Democratic Republic JANE Z. SANDERS (19), Division of Biology, California Institute of Technology, Pasa- dena, California 91125 JON R. SAUNDERS (2), Department of Mi- crobiology, University of Liverpool, Liv- erpool L69 3BX, England IRA SCHILDKRAUT (3, 6), New England Biolabs, Inc., 32 Tozer Road, Beverly, Massachusetts 01915 NOBUKO SHINDO-OKADA (23), Biology Divi- sion, National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104, Japan KAREN SILVERSTEIN (30), Department of Mathematical Sciences, Memphis State University, Memphis, Tennessee 38152 CASSANDRA L SMITH (28), Departments of Microbiology and Psychiatry, College of Physicians and Surgeons of Columbia University, New York, New York 10032 LLOYD M. SMITH (19), Division of Biology, California Institute of Technology, Pasa- dena, California 91125 KEVIN STRUHL (34, 35), Department of Bio- logical Chemistry, Harvard Medical School, Boston, Massachusetts 02115 DIETRICH STUEBER (26), Central Research Units, Hoffman-La Roche and Company AG, CH-4002 Basel, Switzerland THOMAS D. TULLIUS (33), Department of Chemistry, The Johns Hopkins Univer- sity, Baltimore, Maryland 21218 BIK-KWOON TYE (32), Section of Biochem- istry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 GUIDO VOLCKAERT (17), Rega Institute, Faculty of Medicine, University of Leuven, B-3000 Leuven, Belgium JEREMY N. B. WALKER (2), Amersham In- ternational plc, Little Chalfont, Bucking- hamshire HP7 9LL, England K. M. WESTON (7), Medical Research Council Laboratory of Molecular Biol- ogy, Cambridge CB2 2QH, England RAY WU (16), Section of Biochemistry, Mo- lecular and Cell Biology, Cornell Univer- sity, Ithaca, New York 14853 ROBERT ZAGURSKY (11), E. I. du Pont de Nemours & Company, Inc., Central Re- search and Development Department, Experimental Station, Wilmington, Dela- ware 19898 [1] RESTRICTION ENZYME HgiCI 3 [1] Restriction Enzyme HgiCI Characterization of the 6-Nucleotide Staggered Cut Sequence and Its Application in Mismatch Cloning By MANFRED KROGER and GERD HOBOM Thanks to the availability of the rich collections of Drs. Reichenbach 1 and Brown, 2 the gliding bacterium Herpetosiphon giganteus became one of the most intensively screened groups of organisms in the search for new restriction enzymes. Among the 10 strains tested, 17 enzymes could be found with seven different but related recognition sequences. This led to a hypothesis regarding the evolutionary relationship among these en- zymes and could be a basis for a better understanding of the biochemical mechanism of restriction enzyme-DNA target interaction. 3 Among these enzymes HgiCI is remarkably different from all other previously described endonucleases, since it produces 5'-hexanucleotide protruding ends. Combined with the fact that HgiCI recognizes a degen- erated sequence, specific applications of this enzymatic activity in gene technology are possible. Usually, for specific base pairing within 5'- or 3'- protruding ends, a match of 2 bp is fair, while four matching base pairs lead to highly efficient ligase reactions. Since a perfect match of 6 bp may not be required, we used HgiCI-restricted DNA fragments in order to test whether DNA ligase reactions among hexanucleotide protruding ends could proceed in spite of some mismatch positions. Our results presented here allow the conclusion that it is possible to obtain mismatched ligase reaction products in considerable fractions. A wider application of this observation seems possible, since an isoschizomer of HgiCI BanI, is available commercially and is obtained from an unrelated strain Bacillus aneurinolyticus (IAM 1077). In contrast to the data given in the litera- ture, 4 we have determined via cross-ligation that BanI also produces 5'- hexanucleotide protruding DNA fragments. In this article we intend to focus on the methodology used to charac- terize recognition sequences and on the application of HgiCI (BanI) frag- ment ends in mismatch cloning rather than on enzyme purification proce- dures. H. Mayer and H. Reichenbach, J. Bacteriol. 136, 708 (1978). 2 N. L. Brown, M. McClelland, and P. R. Whitehead, Gene 9, 49 (1980). 3 M. Kr6ger, G. Hobom, S. Schiatte, and H. Mayer, Nucleic Acids Res. 12, 3127 (1984).~ 4 I. Schildkraut, cited in R. J. Roberts, Nucleic Acids Res. 12, r167 (1984). Copyright © 1987 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 155 All rights of reproduction in any form reserved. 4 RESTRICTION ENZYMES [1] Purification of HgiCI Herpetosiphon giganteus strain Hpg9 has been obtained from Dr. H. Reichenbach (Gesellschaft fiir Biotechnologische Forschung, Braun- schweig-St6ckheim, Federal Republic of Germany). The conditionally anaerobic strain was grown at 30 ° as described by Mayer and Reichen- bach. ~ After centrifugation at 24,000 g for 15 min at 5 °, 10 g of cells was used in a standard purification procedure, 5 which involved breaking the cells through a Branson Sonifier followed by a single centrifugation step (30 min, 45,000 g). The supernatant was used for column chromatography on DEAE-cellulose DE-52 (2.6 x 15 cm) without any further treatment. The appropriate restriction enzyme-containing fraction was obtained through gradient elution from 0 to 0.3 M NaC1 in l0 mM potassium phos- phate (pH 7.5), 1 mM EDTA, 0.1% (v/v) 2-mercaptoethanol, and 10% glycerol. Restriction endonucleolytic activity was assayed for every frac- tion by incubation with h DNA or some other substrate. Fractions with identical activities were pooled and dialyzed against the buffer given above. The dialyzed enzyme solution was rechromatographed on a phos- phocellulose P11 column (2.6 x 15 cm). NaCI-Dependent elution yields three different restriction enzymes named according to the order of elu- tion: HgiCI, HgiCII, and HgiCIII. A more detailed description of the purification procedure is given by Kr6ger et al. 3 Recognition Sequence Determination The purified enzymes were used to generate a series of fragmentation patterns from completely sequenced plasmid DNAs. Incubation was gen- erally for 2 hr at 37 ° in 10 mM MgCI2 and 10 mM Tris. HC1, pH 7.5. The patterns obtained after agarose gel electrophoresis usually provided enough information to distinguish between cleavage reactions already known and new digestion specificities. Within the H. giganteus strain Hpg9 (C) we could identify HgiCII as an isoschizomer of AvaII(GGT/ ACC) and HgiCIII as an isoschizomer of SatI (G/TCGAC). However, HgiCI digestion resulted in an unknown pattern that could be resolved by double digestions with other enzymes as described in detail by Kr6ger et al. 3 The HgiCI recognition sequence was finally identified as the degener- ated GGPyPuCC sequence. Determination of the Cleavage Site for HgiCI and BanI In principle, two strategies were used to identify the endonucleolytic cleavage sites relative to the respective recognition sequences: (1) chemi- v. Pirrotta and T. A. Bickl¢, this series, Vol. 65, p. 89. [1] RESTRICTION ENZYME HgiCI 5 cal characterization by determination of the 5' nucleotide(s) plus size determination of denatured DNA fragments resulting from enzymatic di- gestion in comparison to a sequencing ladder for that DNA segment, and (2) mixed ligase reaction between restriction fragments obtained after cleavage with two different enzymes. The latter procedure is applicable for any (suspected) isoschizomers or for enzymes producing fragments with identical cohesive ends. In the case of isoschizomers, additional confirmation can be provided by recutting the interligation products with either of the two enzymes. The chemical characterization of the endonucleolytic cleavage posi- tion has been applied for HgiCI as the first enzyme discovered with the recognition sequence GGPyPuCC. An initial determination of the nature of the 5'-terminal nucleotide for several different HgiCI fragments com- prising a full representation of the pyrimidine and purine degeneracies at the two central positions resulted in a G residue as the 5'-terminal nucleo- tide (93 to 95% pG). For this determination of the 5'-terminal nucleotide we used paper electrophoresis, after the unlabeled 5'-phosphate group was changed en- zymatically into a 32p-labeled 5'-phosphate group using alkaline phospha- tase and T4 polynucleotide kinase, following the Maxam-Gilbert proto- col. 6 Usually the 32p-labeling procedure was performed using a mixture of DNA fragments produced from the same plasmid DNA. In order to obtain fragments with only a single labeled end, the primary digests were con- verted into a mixture of subfragments by secondary restriction enzyme digestion prior to isolation. Only those fragments known to contain a single 32p-labeled HgiCI end were isolated and treated further to identify the labeled nucleotide. Each fragment was digested completely into mononucleotides within a volume of 30/zl containing 10 mM Tris (pH 8.5) and 10 mM MgCI2 plus 1/zg DNase I and 1 ~g snake venom phosphodies- terase for 1 hr at 37 °. Then 40/zl of a mixture of unlabeled mononucleo- tides (about 20 mg/ml each) was added as cartier to achieve optical visibil- ity. As described by Kr6ger and Singer 7 the reaction mixture was applied onto Whatman 3MM paper and the mononucleotides were separated in a Savant paper electrophoresis system using 0.12 M ammonium formate buffer, pH 3.5 (2.1 g ammonium formate and 3.3 ml formic acid/liter). The four mononucleotide spots observable on the dry paper sheet were cut out under UV light and were used directly for measuring their 32p activities. The order of separation at pH 3.5 was pC, pA, pG, and pT. Application of a high-performance liquid chromatography (HPLC) separation technique may be recommended as a more modern alternative, especially since the A. M. Maxam and W. Gilbert, this series, Vol. 65, p. 499. 7 M. Krrger and B. Singer, Biochemistry 18, 3493 (1979). 6 RESTRICTION ENZYMES [1] Savant paper electrophoresis requires huge amounts of inflammable pe- troleum. Since this result does not lead to an unambiguous interpretation for the cleavage position, due to the two G residues within the GGPyPuCC HgiCI recognition sequence, a second identification procedure had to be applied. For molecular-weight determination, use has been made of an HgiCI cleavage product gel electrophoretic sizing against a DNA sequencing ladder of a DNA fragment which contains within its known sequence a single cleavage site for HgiCI. For this purpose endonucleolytic cleavage by HgiCI prior to isolation of the desired subfragment was used on part of the terminally labeled material, while the main fraction was converted into a DNA sequencing ladder following the conventional Maxam-Gilbert procedure. The HgiCI cleavage product(s) of the same fragment were loaded in a fifth lane of the sequencing gel. To ensure identical ionic conditions when all five samples are applied onto the acrylamide gel, the HgiCI endonucleolytic digest had been subsequently treated with phenol to remove all of the protein, and precipitated with alcohol in the presence of tRNA. For the correct assignment of the resulting electrophoresis pattern, it is necessary to take into consideration the presence of a phosphate group at the 3' end of each of the chemical fragmentation products. Endonu- cleolytically derived fragments, however, contain free 3'-OH groups. This results in a shift in electrophoretic mobility as shown in Fig. 1. The given assignment indicates that restriction enzyme-generated fragments migrate a shorter distance than the 3'-phosphorylated counterparts of identical chain length. As a control experiment a similar fragmentation reaction has been performed for the well-established Sau3A endonu- cleolytic cleavage site, as is shown in the lower part of Fig. 1. Here we used a DNA fragment with both ends labeled, a smaller part of which was digested with Sau3A, while the main part was used for strand separation on a denaturating acrylamide gel 6 and for DNA sequencing. Thus sizing was possible using two Maxam-Gilbert sequencing ladders for both com- plementary cleavage products, although only an unseparated fragment mixture was used after Sau3A digestion. In light of the two results we were able to show by this procedure that HgiCI produces a hexanu- cleotide staggered cut with an extended 5' terminus, the first enzyme to be observed with this fragmentation characteristic. Mixed ligase reactions are a quick and easy method to characterize an endonucleolytic cleavage reaction, if a suspected isoschizomer is avail- able. We have applied this technique for the AvalI/HgiCII cleavage site and used gel electrophoretic separation to identify the multiple ligation [1] RESTRICTION ENZYME HgiCI 7 HgiCI CT.CA G E Sau3A -GCATCAC, - GCATCAC~ -GCATCAC~ - GCATCAp ~ -GCATC- / ,i., -GCATp ~ -GCAp ~i -GCp ~~ -Gp ~ im -GCATCACCoH G AT.CC E C ToCA G E - GTGGATTG ~ ~" ~ I -GTGGATTp ~ , ~ , - GTGGATp. ~., - GTGGAp - GTGGATToH I -GTGp ___~__.~ ~ ~ I I -% ~" I I -CTGo" j j J FIG. 1. Determination of cleavage sites for endonucleases ItgiCI and Sau3A through gel electrophoretic sizing of cleavage products. Three Maxam-Gilbert DNA sequencing ladders are produced, each serving as a molecular-weight marker series for the respective fragment cleaved either by HgiCI (upper panel) or by Sau3A (lower panels). The terminal fraction of the DNA sequences corresponding to the autoradiographically visible bands is notified at the left margin. The main structural difference between the chemically and the endonucleolyti- caily cleaved products is the absence of the 3'-phosphate group from the fragment in the E lane (E = enzyme treated). In all three cases this leads to an electrophoretic mobility decreased by almost one unit for a pair of the otherwise identical fragments. products in an agarose gel system (for details, see Ref. 3). An alternative to tracing the (multiple) reaction products on gels is provided by determi- nation of cloning frequencies for the respective (co)ligation products. This technique was applied in a coligation analysis for BanI/HgiCI cleavage sites. Both enzymes have been reported to recognize the same degener- ated sequence GGPyPuCC, but BanI cleavage reaction has been de- scribed to result in a four-nucleotide rather than a six-nucleotide stag- gered cut as observed for ngiCI. 4'8 We wanted to redetermine the 8 H. Sugisaki, Y. Maekawa, S. Kanazawa, and M. Takanami, Nucleic Acids Res. 10, 5747 (1982). 8 RESTRICTION ENZYMES [1] cleavage position for the endonuclease BanI relative to HgiCI and have used a mixed ligation procedure as outlined in Fig. 2. In order to provide an easier characterization of the resulting clones we chose two different but closely related plasmids with two HgiCI/BanI sites within two regions of identical sequence. One BanI/HgiCI recogni- tion site was located within the ampicillin resistance gene, thus clones were expected only after successful ligation. The replication function was supplied by one HgiCI fragment only, and the complementing BanI frag- ment contained two extra landmark restriction sites (SphI and SnaBI). (For experimental details, see Fig. 2.) The cloning yield was excellent and all 12 clones analyzed showed the correct restriction pattern for the calcu- lated coligation product, using SphI and BanI for characterization. Thus BanI and HgiCI cleave at identical positions and are true isoschizomers. Eco., .<-, 2 .,n0,,, ®".n ~GGCGCC*AGG~IGGTGCC FIG. 2. Cloning strategy used for a mixed ligase reaction between fragments derived from HgiCI-restricted plasmid pHK255 and the closely related BanI-restricted plasmid pHK402. Plasmid pHK255 is digested first with EcoRI and HindlII and subsequently treated with alkaline phosphatase to remove 5'-phosphate groups from these termini, and the mixture is finally cleaved with HgiCI. The resulting four fragments are without isolation, mixed, and coligated with the isolated BanI fragment, which contained no origin of replication. Using ampiciUin selection the coligation product plasmid pHK422 was obtained exclusively (12 out of 12 clones analyzed) and in high yield. Both analysis by SphI and BanI digestion led to two fragments each (the third BanI/HgiCI site is methylated in Escherichia coli and thus is not cleaved), as expected for the given map of pHK422. [...]... buffer: 100 mM Tris (pH 8.0) 10 mM EDTA l mM EGTA 10/zg/ml BSA (nuclease free) 5 raM dithiothreitol (0-100 mM NaCI) 2 Replace buffer with two volumes of fresh buffer Methylation is performed in a volume twice that of the beads in buffer containing 80/xM SAM and at least 5 U of methylase per microgram of DNA Incubation is performed for 2 hr, and then the buffer is changed and the procedure repeated for... Introduction Site-specific DNA methylases (S-adenosylmethionine : DNA methyltransferases) have a variety of uses in DNA analytical procedures Methylases may be used as probes for DNA conformation,L2 in recombinant DNA cloning strategies, 3 or in combination with restriction endonucleases to generate rare or novel DNA cleavage specificities 4-9 DNA methylases have been purified from a number of different sources... glycerol The enzyme is stored at - 2 0 ° The final yield of SfiI is 1000 U per gram of cells Of the SfiI preparation, 50 U releases less than 0.3% of radioactivity in the exonuclease assay, and 50 U of the SfiI preparation converts less than 10% of tbX174 RF I DNA to RF II in the nonspecific endonuclease assay Properties of NotI and S//I SfiI and N o d are classified as type II restriction cndonucleases... The electrophoresis buffer is 90 mM Tris, 90 mM borate, 1 mM EDTA, and 0.5/zg/ml ethidium bromide The resulting DNA banding pattern is visualized by ethidium bromide fluorescence under short-wave ultraviolet light The sizes (base pairs) of the eight DNA fragments produced by NotI cleavage of Adeno-2 DNA and the sizes of the four DNA fragments produced by SfiI cleavage of Adeno-2 DNA are listed in Table... number of commercially available Type II DNA methylases, using the M.MbolI methylase purification as an instructive example) These assay procedures have been successfully applied to several different DNA methylases possessing four-, five-, six-, and eight-nucleotide sequence specificities Materials Restriction endonucleases, purified DNA methylases, T4 DNA ligase, unmethylated phage h DNA, ~bX174 RF I DNA, ... protect bacteriophage DNA from cleavage by a particular restriction endonuclease Such methods are adequate in some cases, and purified DNA methylases are defined in terms of protection units However, a more sensitive method has been successfully used on a commercial scale for the purification of over 20 different DNA methylases In this assay a synthetic duplex oligonucleotide is self-ligated (polymerized)... 2 0 ° The final yield of NotI is 500 U per gram of cells Of the NotI preparation, 10 U releases 0.5% of radioactivity in the exonuclease assay, and 25 U of the NotI preparation converts less than 10% of thX174 RF I DNA to RF II in the nonspecific endonuclease assay Purification of S/iI Streptomyces fimbriatus ATCC 15051 is grown at 30° to late logarithmic phase in a medium consisting of I0 g/liter... replaced twice with restriction buffer (vendor's recommended conditions) DpnI buffer is as follows: 150 mM NaC1 100 mM Tris HCI (pH 8.0) 10 mM MgC12 100/zg/ml BSA 2 Digestion is performed in twice the bead volume using 5 U of DpnI/ /xg DNA for 3 hr at 37° with shaking It is usually advisable then to change the restriction buffer and redigest for a further 3 hr DpnI is available from New England Biolabs and... electrophoresis, one layer of a doubledecker gel can be stained for UV visualization, dried for autoradiography, or blotted to nitrocellulose while the other identical layer remains as a source of intact DNA This method is of particular use for PFG because of the variability in PFG DNA separation patterns even for gels run under ostensibly identical conditions Double-decker gels are made as follows: 1 Melted... identical DNA pattern can be produced at the first attempt Applications include identifying a DNA of interest in one layer then aligning this information with the second layer and obtaining the DNA intact from this layer 1 The position of a band of interest can be determined from an ethidium-stained layer on a UV box The DNA band is then cut from the other layer This band will be ethidium free and has . defined as that amount of enzyme which completely digests 1 gg of Adeno-2 DNA in 1 hr at 50 ° in 50/zl of SfiI buffer. The maximum rate of cleavage is obtained at 50 °. The activity is 10-fold. of ~bX174 RF I DNA in the standard restriction endonuclease assay buffer in a volume of 0.05 ml. The supercoiled ~bX174 DNA has been substituted for Adeno-2 DNA because neither NotI nor SfiI. a match of 2 bp is fair, while four matching base pairs lead to highly efficient ligase reactions. Since a perfect match of 6 bp may not be required, we used HgiCI-restricted DNA fragments