Polymerases Enzyme Resource Guide

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Polymerases Enzyme Resource Guide

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1 Enzyme Resource Guide Polymerases from Promega Enzyme Resource Guide Polymerases from Promega MADE IN WISCONSIN Promega’s high quality products are manufactured in Madison, Wisconsin, and distributed worldwide. We often say we are a “fully-integrated” manufacturing facility. But what does that mean? For enzyme production, it means a lot! We have an extensive culture collection, maintained by cryopreservation and backed up by off-site redundant storage. Our advanced fermentation capabilities range from small volumes to thousands of liters, all carefully monitored and controlled by a state-of-the-art programmable logic controller (PLC). At each step of the biomass production, from removal of a frozen vial from the master seed collection to cell harvesting with the continuous flow centrifuges, quality control is diligently maintained through assaying for pure culture and product. We use sophisticated purification techniques to produce enzyme fractions from crude cell extracts. Whether an enzyme was produced last year or will be produced next year, Promega ensures consistency through process design and clear and concise purification protocols. We use process equipment designed to operate at large scale, such as tangential flow filtration, which rapidly separates contaminants from the process stream. These rapid processing steps maximize enzyme quality while providing better value to our customers by reducing costs. Thorough assays are used both throughout and after the purification process. Enzymes are tested for activity, specific activity, contaminating nucleases, and specific performance characteristics before being dispensed, labeled, packaged, and stored in specially designed freezers. Automation of these processes speeds workflow and increases accuracy. Promega enzymes are shipped rapidly through our worldwide distribution network to meet your need for high-quality, readily available enzymes. We don’t stop there – Promega’s Technical Services Scientists are ready to respond to customer questions by telephone, fax, or email. In order to continually improve quality and service to our customers, dedicated Customer Focus Teams review customer feedback, manufacturing processes, training programs, new product concepts and technical support materials. That’s what we mean by “fully-integrated”; an organization committed to providing quality and value to our customers worldwide. Promega earned ISO 9001 registration in 1998. This registration assures that quality systems have been designed, implemented and audited in all product development, production, shipping, and support areas, and stands as a visible symbol of our commitment to continual process improvement. ONE i TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com CONTENTS Introduction: The Polymerases ……………………………………………………………………………………………1 DNA Polymerases …………………………………………………………………………………………………………………2 DNA Polymerase I……………………………………………………………………………………………………………3 DNA Polymerase I Large (Klenow) Fragment………………………………………………………………..4 DNA Polymerase I Large (Klenow) Fragment, Exonuclease Minus……………………………..5 T4 DNA Polymerase………………………………………………………………………………………………………..6 Thermophilic DNA Polymerases ………………………………………………………………………………………….9 Taq DNA Polymerase…………………………………………………………………………………………………….10 Sequencing Grade Taq DNA Polymerase…………………………………………………………………….12 Tfl DNA Polymerase………………………………………………………………………………………………………13 Tth DNA Polymerase……………………………………………………………………………………………………..14 Tli DNA Polymerase ……………………………………………………………………………………………………..16 Pfu DNA Polymerase……………………………………………………………………………………………………..17 RNA Polymerases……………………………………………………………………………………………………………….19 T7 RNA Polymerase………………………………………………………………………………………………………20 T3 RNA Polymerase………………………………………………………………………………………………………22 SP6 RNA Polymerase……………………………………………………………………………………………………23 Reverse Transcriptases………………………………………………………………………………………………………25 AMV Reverse Transcriptase………………………………………………………………………………………….26 M-MLV Reverse Transcriptase ……………………………………………………………………………………..28 M-MLV Reverse Transcriptase, RNase H Minus………………………………………………………….29 Terminal Deoxynucleotidyl Transferase ……………………………………………………………………………30 Glossary………………………………………………………………………………………………………………………………34 ENZYME RESOURCE GUIDE POLYMERASES PROMEGA ENZYME RESOURCE GUIDE ii ONE DNA Polymerase a. b. c. d. e. f. g. h. i. Mg2+, 4 dNTPs 3′ 5′ Mg2+, 4 dNTPs Mg2+ G 5′ 3′ 5′ 5′ 3′ 5′ 5′ 3′ DNA Polymerase with Proofreading Activity 3′ 3′ 5′ 5′ 3′ 3′ 5′ 5′ (N)n (N)n 5′ 5′ RNA Polymerase Reverse Transcriptase Terminal Transferase 3′ promoter terminator 3′ 5′ 5′ 5′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ 5′ 3′ 5′ Mg2+, 4 dNTPs Mg2+, 4 rNTPs Mg2+, 4 rNTPs Mg2+, 4 dNTPs Mg2+, 4 dNTPs Metal2+, ndNTP 5′ 5′ 3′ 3′ 3′ 5′ 5′ 3′ 3′ 5′ 5′ 5′ 3′ 3′ 3′ 5′ 5′ 3′ 3′ 3′ 3′ 3′ 5′ 5′ 5′ 5′ 3′ promoter 3′ 5′ 5′ 5′ 3′ 3′ TTT promoter terminator T A Figure 1. Diagram depicting enzymatic activities of polymerases commonly used in molecular biology applications. Straight lines indicate DNA molecules, wavy lines indicate RNA molecules and arrowheads indicate the 3′-end of a nucleic acid molecule. Panel a: DNA polymerases incorporate dNTPs 5′→3′ from the 3′-end of a DNA initiator annealed to ssDNA. Panel b: DNA polymerases possessing a 3′→5′ exonuclease (proofreading) activity can repair nucleotide misincorporations. Panel c: In the presence of dNTPs, polymerases possessing a 3′→5′ exonuclease activity will remove ssDNA 3′-overhangs from dsDNA. Panel d: In the absence of dNTPs, these enzymes will continue to remove 3′-nucleotides from dsDNA. Panels e and f: RNA polymerases incorporate NTPs 5′→3′ on a DNA template from a specific dsDNA promoter. Panel e: Polymerase-specific terminator sequences will terminate transcription, although with less than 100% efficiency. Panel f: Allowing the polymerase to “run-off” the end of the DNA template will produce a higher percentage of identically sized transcripts. Panels g and h: Reverse transcriptases incorporate dNTPs 5′→3′ from the 3′-end of a DNA initiator annealed to RNA (Panel g) or DNA (Panel h). Panel i: Terminal deoxynucleotidyl transferase adds one or more dNTPs to the 3′-end of ds- or ssDNA. promoter ONE 1 ENZYME RESOURCE GUIDE POLYMERASES INTRODUCTION: The Polymerases Enzymes that modify nucleic acids provide the foundation for many of the techniques in molecular biology. These enzymes are used to synthesize, degrade, join or remove portions of nucleic acids in a controlled and generally defined manner. Specific features of the in vivo functions of these enzymes have been exploited in vitro to provide many of the protocols currently used in nucleic acid manipulations. Figure 1 summarizes the activities of polymerases generally used for these purposes. This chapter describes many of the commonly used polymerases and their basic characteristics, with an emphasis on their practical applications in today’s laboratories. While a number of these enzymes may perform the same general function, one enzyme may be a better choice than the others for a specific purpose. This information is imparted in the individual enzyme descriptions and Table 1. The reaction conditions provided in each entry are those optimized at Promega, unless otherwise indicated. Many of the techniques using polymerases are detailed in Promega’s Protocols and Applications Guide, Third Edition, in Promega system protocols (technical bulletins and technical manuals) and in references to these detailed protocols provided within each enzyme description in this guide. Table 1. Recommended Applications of Polymerases. Klenow T4 Taq Seq. Grade Tfl Tth Tli Pfu T7 T3 SP6 MMLV DNA Pol. I Klenow exo minus DNA Pol. DNA Pol. Taq DNA Pol. DNA Pol. DNA Pol. DNA Pol. RNA Pol. RNA Pol. RNA Pol. AMV RT MMLV RT RT H minus TdT Labeling 3′ addition ✓ ✓ ✓ ✓ ✓ ✓ 3′ fill-in ✓ ✓ ✓ ✓ ✓✓✓✓ ✓ ✓ ✓ 3′ replacement ✓ ✓ ✓ ✓ ✓ Nick translation ✓ Replacement ✓ ✓ ✓ ✓ ✓ synthesis Random priming ✓ ✓ RNA probes ✓ ✓ ✓ Cloning Creating blunt ends ✓ ✓ ✓ ✓ 5′-overhang fill-in ✓ ✓ ✓ ✓ ✓ 3′-overhang removal ✓ ✓ ✓ ✓ ✓ Sequencing DNA ✓ ✓ ✓ ✓ ✓ ✓ ✓ RNA ✓ ✓ ✓ Mutagenesis Oligo-directed ✓ ✓ ✓ Misincorporation ✓ ✓ ✓ ✓ Nested deletions ✓ Mapping Transcript ✓ ✓ ✓ cDNA Synthesis 1st strand ✓ ✓ ✓ 2nd strand ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ In vitro ✓ ✓ ✓ transcription PCR(g) ✓ ✓ ✓ ✓ ✓ RT-PCR(g) (one enzyme) ✓ ✓ ✓ Preferred enzyme for this application. ✓ Acceptable enzyme for this application (not preferred). ✓ Acceptable enzyme at elevated temperature (>65˚C). PROMEGA ENZYME RESOURCE GUIDE 2 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com DNA Polymerases DNA polymerases meet the cell’s need for DNA-directed DNA synthesis during processes such as chromosomal replication and DNA repair. DNA polymerases have two substrates: one is the template-bound primer terminus containing a free 3′-hydroxyl (OH) group, the other is a deoxynucleotide 5′-triphosphate (dNTP) (Figure 1a; 1). A phosphodiester bridge is formed by nucleophilic attack of the 3′-OH of the primer terminus on the α-phosphate group of the dNTP and elimination of the terminal pyrophosphate (PPi ): (dNMP)n + dNTP d(NMP)n+1 + PPi DNA polymerases require a primer with a free 3′-OH, otherwise new DNA chains cannot be initiated. The newly added monomer has a free 3′-OH group and can serve as a primer terminus for another round of polymerization. Chain elongation during polymerization occurs in the 5′→3′ direction; polymerization is either distributive or processive. A distributive polymerase dissociates from the template after the addition of each nucleotide, whereas a processive polymerase maintains its association with the template for a succession of nucleotide additions. Some polymerases depend upon accessory factors for their high processivity (2,3). This may be a consideration when using these polymerases in vitro, as commercial preparations may not contain the required cofactors. Another consideration is the reverse reaction, which yields free dNTPs (4). In living cells, the reaction is strongly biased in the forward direction by an enzyme called pyrophosphatase, which hydrolyzes the pyrophosphate (PPi ) to inorganic phosphate. The number of nucleotides added by a DNA polymerase before it dissociates from the primer-template is often expressed as a processivity index or value (5). Although this index can be measured, it is unclear how important processivity is in most molecular biology applications because theoretically saturating concentrations of enzyme are used in most techniques. Other factors to consider are turnover rate and fidelity (6). The turnover rate is the maximum number of nucleotides polymerized per minute per molecule of DNA polymerase. It should not be confused with processivity; the turnover rate does not consider whether a polymerase is processive or distributive. Fidelity is the enzyme’s ability to faithfully replicate a DNA molecule. Error rates are commonly expressed as the mutation rate per base pair duplicated, and accuracy as the inverse of error rate. In other words, accuracy is the average number of nucleotides the polymerase incorporates before making an error. Some DNA polymerases have functions other than that of polymerization. The polymerase may have an associated 3′→5′ exonuclease activity, 5′→3′ exonuclease activity, the ability to use RNA rather than DNA as substrate (reverse transcription) and nontemplate-dependent addition of nucleotides. The 3′-exonuclease activity is associated with a proofreading role, where it removes an incorrectly inserted (noncomplementary) base and thereby allows the polymerase reaction to continue (Figure 1b). The proofreading and polymerization reactions are in competition (Figures 1a, c and d). Generally, a DNA polymerase with 3′-exonuclease activity will demonstrate higher fidelity than one without, which may affect enzyme choice for some applications. The so-called 5′-exonuclease activity is involved with excision repair within a cell (7). This activity is actually that of a single-strand dependent endonuclease, so the conventional name is inaccurate (8). While this activity is necessary for applications such as nick translation (9,10), it is undesirable for DNA sequencing applications (11). Additionally, some DNA polymerases have a reverse transcriptase activity (replication of DNA from RNA templates), which is usually enhanced by altering the cation in the reaction (e.g., Mn2+ instead of Mg2+). DNA polymerases lacking a 3′-exonuclease may add a nontemplate-dependent nucleotide to the 3′-end, behaving, in other words, like a terminal transferase (12). However, unlike terminal transferase, only a single nucleotide is added to the 3′-end. There is a preference for the nucleotide added, often dATP, but this preference is sensitive to the sequence context near the 3′-end (12). Two strategies are used to clone fragments carrying the nucleotide added by nonproofreading polymerases. One is to “polish” or blunt the ends of the DNA fragment, for example, by using a proofreading DNA polymerase. Another approach is to use vector that compensates for the added nucleotide, for example, a T-vector (13). Of the DNA polymerases used for molecular biology manipulations, the most common are those isolated from bacterial or bacteriophage sources. These can be divided into two groups, based on the enzyme’s thermostability (i.e., the ability of the enzyme to withstand prolonged exposure to an elevated temperature). Thermostable DNA polymerases tend to have an optimum reaction temperature of approximately 74˚C, but can withstand repeated exposures to temperatures higher than the optimum. Mesophilic DNA polymerases have optimum reaction temperatures much lower than the thermostable DNA polymerases (usually 37–42˚C) and cannot withstand prolonged exposure to temperatures higher than this optimum. References 1. Kornberg, A. (1969) Science 163, 1410. 2. Selik, H.E. et al. (1987) In: DNA Replication and Recombination, McMacken, R. and Kelly, J.J., eds., UCLA, A.R. Liss, New York, 47, 183. 3. Tabor, S., Huber, H.E., Richardson, C.C. (1986) J. Biol. Chem. 261, 16212. 4. Deutscher, M.P. and Kornberg, A. (1969) J. Biol. Chem., 244, 3019. 5. Kornberg, A. and Baker, T.A. (1992) In: DNA Replication, 2nd edition, W.H. Freeman and Company, New York, 494. 6. Kornberg, A. and Baker, T.A. (1992) In: DNA Replication, 2nd edition, W.H. Freeman and Company, New York, 496. 7. Kornberg, A. and Baker, T.A. (1992) In: DNA Replication, 2nd edition, W.H. Freeman and Company, New York, 140. 8. Lyamichev, V., Brow, M.A.D. and Dahlberg, J.E. (1993) Science 260, 778. 9. Protocols and Applications Guide, Third Edition (1996) Promega Corporation,125. 10. Kornberg, A. and Baker, T.A. (1992) In: DNA Replication, 2nd edition, W.H. Freeman and Company, New York, 142. 11. Protocols and Applications Guide, Third Edition (1996) Promega Corporation, 149. 12. Hu, G. (1993) DNA Cell Biol. 12, 763. 13. Protocols and Applications Guide, Third Edition (1996) Promega Corporation, 41. ONE 3 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com ENZYME RESOURCE GUIDE POLYMERASES DNA Polymerase I E.C. 2.7.7.7 Description DNA Polymerase I (Cat.# M2055) is a DNA-dependent DNA Polymerase that has three activities: 5′→3′ polymerase, 5′→3′ exonuclease and 3′→5′ exonuclease (1,2). DNA Polymerase I is most commonly used in combination with DNase I for generating labeled DNA probes by nick translation (3,4). The enzyme can also be used for the labeling of 3′-ends of DNA by 3′ fill-in or 3′ replacement. DNA Polymerase I can be used to convert 5′- and 3′-overhangs to blunt ends; however, the Klenow Fragment of DNA Polymerase I or T4 DNA Polymerase is preferred for these applications. DNA Polymerase I can be used for DNA sequencing, although it is not the enzyme of choice due to its exonuclease activity. The enzyme can also be used in oligodirected mutagenesis; however, T4 DNA Polymerase is preferred for this application due to the relatively low fidelity and strand displacement activity of DNA Polymerase I. This lower fidelity makes DNA Polymerase I more suitable for mutagenesis by nucleotide misincorporation. DNA Polymerase I is also commonly used in combination with RNase H for second-strand synthesis of cDNA (5). Applications •Nick translation (3,4). •3′-end labeling. •Conversion of 5′- and 3′- overhangs to blunt ends. •Second-strand synthesis of cDNA (5). •Mutagenesis by misincorporation. DNA Polymerase I is a component of the following Promega systems: •Nick Translation System (Cat.# U1001) •Universal RiboClone® cDNA Synthesis System(a) (Cat.# C4360) Enzyme Properties Requirements: Divalent cations are required for both the polymerase and exonuclease activities of the enzyme (2). A concentration of 7mM Mg2+ is optimal (6). Optimal Substrate: For polymerase activity, the optimal substrate is primed ssDNA. DNA Polymerase I will also extend from single-strand nicks or gaps in dsDNA. The enzyme is active on DNA:RNA and RNA:RNA hybrids, under certain conditions (7). The 3′→5′ exonuclease activity of DNA Polymerase I affects both ss- and dsDNA. The 5′→3′ exonuclease activity affects dsDNA and RNA:DNA hybrids. Optimal pH: 7.4 with potassium phosphate buffers (6). Km: For dNTPs, 1–2µM (7). For DNA, 2µM (8). Alternative Cofactors and Substrates: Mn2+ can fulfill the requirement for the divalent cation; however, the activity and fidelity of the enzyme is reduced (6,9). Hapten-labeled nucleotides can be incorporated by DNA Polymerase I (10), including biotinylated (11) and digoxigenin-labeled (12) nucleotides. Inhibitors: Polymerization by DNA Polymerase I is inhibited by a variety of agents including antibiotics, DNA intercalating and alkylating agents and nucleotide analogs (2,13). Extinction Coefficient/Absorbance: The absorbance of a 1mg/ml solution in 10mM sodium bicarbonate at 280nm is 0.85 (14). Strand Displacement Activity: Yes. Inactivation: 68˚C for 10 minutes (16). Error Rate: 9 x 10–6 (15). Genetic Locus: E. coli polA. Promega Product Information Source: DNA Polymerase I is purified from the recombinant E. coli strain CM5199 (17), which is a lysogen carrying a lambda polA transducing phage. Molecular Weight: E. coli DNA Polymerase I is a monomeric protein first described as 109kDa in size (14). However, as deduced from the sequence of the polA gene of E. coli, the enzyme has a calculated molecular weight of 103kDa (18). Storage Conditions: Store at –20˚C. Avoid exposure to frequent temperature changes. DNA Polymerase I is supplied in storage buffer containing 50mM Tris-HCl (pH 7.5 at 25˚C), 1mM DTT, 0.1mM EDTA and 50% (v/v) glycerol. Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 10nmol of deoxyribonucleotides into TCA-insoluble form in 30 minutes at 37˚C. For unit definition the reaction conditions are: 67mM potassium phosphate (pH 7.5), 6.7mM MgCl2, 1mM DTT, 50µg/ml activated calf thymus DNA and 33µM of each dNTP. Purity: The purity is >90% as judged by SDS-polyacrylamide gel electrophoresis with Coomassie® blue staining. Contaminant Assay Endonuclease Assay: To test for endonuclease activity, 1µg of Type I supercoiled plasmid DNA is incubated with 25 units of DNA Polymerase I for 5 hours at 37˚C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting. References 1. Lehman, R. (1981) In: The Enzymes, Vol. XIV, Part A, Boyer, P.D., ed., Academic Press, San Diego, California. 2. Kornberg, A. and Baker, T. A. (1992) DNA Replication, W.H. Freeman and Company, San Francisco, California. 3. Rigby, P.W. et al. (1977) J. Mol. Biol. 113, 237. 4. Nick Translation System Technical Bulletin #TB044, Promega Corporation. 5. Gubler, U. and Hoffman, B.J. (1983) Gene 25, 263. 6. Richardson, C.C. et al. (1964) J. Biol. Chem. 239, 222. 7. Setlow, P. (1974) Meth. Enzymol. 29, 3. 8. McClure, W.R. and Jovin, T.M. (1975) J. Biol. Chem. 250, 4073. 9. Tabor, S. and Richardson, C.C. (1989) Proc. Natl. Acad. Sci. USA 86, 4076. 10. Kessler, C. (1992) In: Nonisotopic DNA Probe Techniques, Kricka, L.J., ed., Academic Press, San Diego, California. 11. Langer, P.R. et al. (1981) Proc. Natl. Acad. Sci. USA 78, 6633. 12. Holtke, H.J. et al. (1990) Biol. Chem. Hoppe-Seyler 371, 929. 13.Zollner, H. (1993) In: Handbook of Enzyme Inhibitors, Second Edition, Part A, VCH, Weinheim. 14. Jovin, T.M. et al. (1969) J. Biol. Chem. 244, 2996. 15. Kunkel, T.A. et al. (1984) J. Biol. Chem. 256, 1539. 16. Perbal. B. (1988) A Practical Guide to Molecular Cloning, Second Edition, John Wiley and Sons, New York. 17. Kelley, W.S. and Stump, K.H. (1979) J. Biol. Chem. 254, 3206. 18. Joyce, C.M. et al. (1982) J. Biol. Chem. 257, 1958. PROMEGA ENZYME RESOURCE GUIDE 4 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com DNA Polymerase I Large (Klenow) Fragment E.C. 2.7.7.7 Description The Large (Klenow) Fragment of DNA Polymerase I (Cat.# M2206) is a DNAdependent DNA polymerase that lacks the 5′→3′ exonuclease activity of intact E. coli DNA polymerase I, but retains the 5′→3′ polymerase, 3′→5′ exonuclease and strand displacement activities. The Large (Klenow) Fragment of DNA Polymerase I is used in several applications including dideoxy sequencing (1), second-strand cDNA synthesis (2) and random primer labeling (3–5). The enzyme can also be used to fill 5′-overhangs with labeled or unlabeled dNTPs (6). The 3′→5′ exonuclease activity of the enzyme can be used to generate blunt ends from a 3′- overhang (7); however, the stronger 3′→5′ exonuclease activity of T4 DNA Polymerase makes it the enzyme of choice for this application (8). The Large (Klenow) Fragment of DNA Polymerase I is also used for in vitro mutagenesis experiments using synthetic oligonucleotides (9). One problem with using the Klenow Fragment for this application is that the strand displacement activity of the enzyme tends to displace the mutagenic primer, thereby removing the intended mutation. As such, T4 DNA Polymerase, which has no strand displacement activity, is a superior enzyme for this application. Applications • Dideoxy sequencing (1). • Second-strand cDNA synthesis (2). • Random primer labeling (3–5). Enzyme Properties Requirements: Mg2+. Optimal Substrate: For 5′→3′ polymerase activity, the optimal substrate is a DNA primer with a 3′-OH terminus annealed to ssDNA. The enzyme will also extend from a nick or gap in double-stranded DNA but will not translate the nick due to the enzyme’s lack of 5′→3′ exonuclease activity. For 3′→5′ exonuclease activity, the optimal substrate is ss- or dsDNA with a 3′-OH terminus. Optimal pH: 7.4 (phosphate), 8.4 (Tris) (11). Km: For dNTPs, 2µM (10). Inhibitors: DNA Polymerase I Large (Klenow) Fragment is inhibited by the adenosine analog adenosine-2′, 2′-riboexpoxide 5′- triphosphate (12). Figures 2, 3 and 4 provide information on the effects of various restriction enzyme buffers, salt concentrations and temperatures, respectively, on DNA Polymerase I Large (Klenow) Fragment. Inactivation: 75˚C for 10 minutes. Error Rate: 4 x 10–5 (13). Strand Displacement Activity: Yes. Genetic Locus: E. coli polA. Promega Product Information Source: Purified from an E. coli strain expressing a recombinant clone (14). Molecular Weight: The Large (Klenow) Fragment of DNA Polymerase I is a 68kDa monomer. The enzyme is a C-terminal fragment of DNA Polymerase I (103kDa). Typical Working Conditions: For optimal activity, use the Klenow 10X Buffer supplied with the enzyme (Table 12). DNA Polymerase I Large (Klenow) Fragment is also active in many restriction enzyme buffers; some users may choose to perform the fill-in reaction directly in the restriction buffer. For other applications, such as sequencing, the optimal reaction conditions are described in the Promega Product Information sheet provided with the enzyme. Storage Conditions: Store at –20˚C. DNA Polymerase I Large (Klenow) Fragment is supplied in storage buffer containing 50mM Tris-HCl (pH 7.5 at 25˚C), 1mM DTT, 0.1mM EDTA and 50% (v/v) glycerol. Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 10nmol of total deoxyribo-nucleotides into TCA-insoluble form in 30 minutes at 37˚C. For unit definition the reaction conditions are: 67mM potassium phosphate (pH 7.5), 6.7mM MgCl2 , 1mM DTT, 50µg/ml activated calf thymus DNA and 33µM each of dATP, dCTP, dGTP and radiolabeled dTTP. Purity: The DNA Polymerase I Large (Klenow) Fragment preparation is >90% pure by SDSpolyacrylamide gel electrophoresis. Klenow Fragment is free from intact DNA polymerase and small fragment, as indicated by SDSpolyacrylamide gel electrophoresis. Contaminant Assay Endonuclease Assay: To test for endonuclease activity, 1µg of Type I (supercoiled) plasmid DNA is incubated with 25 units of DNA Polymerase I Large (Klenow) Fragment in Klenow 1X Buffer (Table 12) for 16 hours at 37˚C. Following incubation, the DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting. References 1. Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463. 2. Houdebine, L.M. (1976) Nucl. Acids Res. 3, 615. 3. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6. 4. Feinberg, A.P. and Vogelstein, B. (1984) Anal. Biochem. 137, 266. 5. Prime-a-Gene ® Labeling System Technical Bulletin #TB049, Promega Corporation. 6. Anderson, S. et al. (1980) Nucl. Acids Res. 8, 1731. 7. Tabor, S. and Struhl, K. (1987) In: Current Protocols in Molecular Biology, F.M. Ausubel et al. eds., John Wiley and Sons, New York. 8. Burd, J.F. and Wells, R.D. (1974) J. Biol. Chem. 249, 7094. 9. Wallace, R.B. et al. (1980) Science 209, 1396. 10.Polesky, A.H. et al. (1990) J. Biol. Chem. 265, 14579. 11.Sambrook, J. et al. (1989) In: Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 12.Abboud, M.M. et al. (1978) J. Biol. Chem. 253, 3415. 13.Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008. 14.Joyce, C.M. and Grindley, N.D. (1983) Proc. Natl. Acad. Sci. USA 80, 1830. ONE 5 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com ENZYME RESOURCE GUIDE POLYMERASES ABCDEGH 0 20 40 60 80 100 120 140 Activity (percent control) Restriction Enzyme Buffer T4 DNA Polymerase DNA Polymerase I (Large) Klenow Fragment DNA Polymerase I (Large) Klenow Fragment, Exonuclease Minus Figure 2. Activity of DNA polymerases in Promega Restriction Enzyme Core Buffers (see also Table 11). Control activity was determined for each polymerase using its standard reaction conditions, buffer composition and temperature, specified for Unit Definition. References 1. Derbyshire, V. et al. (1988) Science 240, 199. 2. Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463. 3. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6. 4. Feinberg, A.P. and Vogelstein, B. (1984) Anal. Biochem. 137, 266. 5. Walker, G.T. (1993) PCR Meth. Appl. 3, 1. 6. Clark, J.M. et al. (1987) J. Mol. Biol. 198, 123. 7. Joyce, C.M. and Grindley, N.D. (1983) Proc. Natl. Acad. Sci. USA 80, 1830. DNA Polymerase I Large (Klenow) Fragment, Exonuclease Minus E.C. 2.7.7.7 Description The Large (Klenow) Fragment, Exonuclease Minus, of DNA Polymerase I (Cat.# M2185) is a DNA-dependent DNA polymerase that lacks both the 5′→3′ and 3′→5′ exonuclease activities of intact E. coli DNA polymerase I (1,2). The enzyme is primarily used for dideoxy sequencing (3). It is also used for random primer labeling (4,5) and in strand displacement amplification (SDA) (6). Klenow Fragment, Exonuclease Minus, will leave a single-base 3′-overhang on a significant proportion of DNA fragments during fill-in of 5′-overhangs (7). Therefore, this enzyme is not recommended for preparation of bluntend fragments for ligation. Applications • Dideoxy sequencing (3). • Random primer labeling (4,5). • Strand displacement amplification (6). Enzyme Properties Requirements: Mg2+. Optimal Substrate: The enzyme is active on a DNA primer having a 3′-OH terminus annealed to ssDNA. Inhibitors: Figures 2, 3 and 4 provide information on the effects of various restriction enzyme buffers, salt concentrations and temperatures, respectively, on DNA Polymerase I Large (Klenow) Fragment, Exonuclease Minus, activity. Inactivation: 75˚C for 10 minutes. Promega Product Information Source: DNA Polymerase I Large (Klenow) Fragment, Exonuclease Minus, is purified from an E. coli strain expressing a recombinant clone (7). Molecular Weight: 68kDa. Storage Conditions: Store at –20˚C. DNA Polymerase I Large (Klenow) Fragment, Exonuclease Minus, is supplied in storage buffer containing 50mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA and 50% (v/v) glycerol. Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 10nmol of deoxynucleotide into acid-precipitable material in 30 minutes at 37˚C. For unit definition the reaction conditions are: 67mM potassium phosphate (pH 7.5), 6.7mM MgCl2, 1mM DTT, 50µg/ml activated calf thymus DNA and 33µM each of dCTP, dATP, dGTP and [3H]dTTP. Purity: The purity is >90% as judged by SDSpolyacrylamide gel electrophoresis with Coomassie® blue staining. Contaminant Assays Endonuclease Assay: To test for endonuclease activity, 1µg of Type I (supercoiled) plasmid DNA is incubated with 25 units of Klenow Fragment, Exonuclease Minus, in Klenow 1X Buffer for 16 hours at 37˚C (Table 12). Following incubation, the DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting. Exonuclease Assay: To test for DNase activity, 50ng of radiolabeled ssDNA and dsDNA are incubated with 30 units of Klenow Fragment, Exonuclease Minus, in Klenow 1X Buffer (Table 12) for 16 hours at 37˚C. The release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is <1% release for ssDNA and <2% release for dsDNA. PROMEGA ENZYME RESOURCE GUIDE 6 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com T4 DNA Polymerase E.C. 2.7.7.7 Description T4 DNA Polymerase (Cat.# M4211 and M4215) is a DNA-dependent DNA polymerase that catalyzes the repetitive addition of nucleotide triphosphates to the 3′-OH of a DNA initiator (i.e., primer) bound to ssDNA. T4 DNA Polymerase also possesses a potent 3′→5′ exonuclease activity on both ssDNA and dsDNA templates (1,2). T4 DNA Polymerase is the highest fidelity polymerase available. T4 DNA Polymerase can be used for labeling of 3′ ends of DNA by 5′ fill-in or the 3′ replacement or exchange reaction (1,3). The enzyme is also commonly used for fill-in of 5′-overhangs and removal of 3′-overhangs prior to ligation (1,3). T4 DNA Polymerase is preferred over DNA Polymerase I Large (Klenow) Fragment for the conversion of 3′-overhangs to blunt ends because of the superior 3′→5′ exonuclease activity of T4 DNA Polymerase. T4 DNA Polymerase is also the enzyme of choice for site-directed mutagenesis, because of the enzyme’s high fidelity and lack of strand displacement activity (1,3). Applications • Polishing or fill-in of 5′-protruding ends with labeled or unlabeled dNTPs (1,3). • Blunting 3′-overhangs (1,3). • Labeling 3′-termini of DNA fragments (replacement reaction) (1,3). • In vitro mutagenesis (1,3). T4 DNA Polymerase is a component of the following Promega systems: •Subcloning Applications Pack (Cat.# M7670) •Universal RiboClone® cDNA Synthesis System(a) (Cat.# C4360) •Altered Sites® II in vitro Mutagenesis System(b) (Cat.# Q5590, Q6600, Q6511, Q6080, Q6090, Q6501, Q6210, Q6491) •GeneEditor™ in vitro Site-Directed Mutagenesis System(c) (Cat.# Q9280) Enzyme Properties Requirements: Mg2+. Cofactor Concentration: Mg2+, 6mM optimal (6). Optimal Substrate: Polymerase activity: primed ssDNA. T4 DNA Polymerase will fill a gap in dsDNA but will not extend from a nick. Exonuclease activity: ssDNA and dsDNA. Typical Working Concentration: For most applications, use approximately five units T4 DNA Polymerase per microgram DNA and 100µM dNTPs (5). Optimal pH: 8.0–9.0 (50% activity at pH 7.5 and pH 9.7) (6). Km: For dNTPs, 2µM (4). Stimulators: Two-fold stimulation at 1mM spermidine. Approximately 25% stimulation at 50mM sodium acetate*. Approximately 20% stimulation at 100mM ammonium acetate. T4 DNA Polymerase activity is increased in the presence of the T4 DNA Polymerase accessory proteins T4 Gene 45 protein and the T4 Gene 44/62 protein complex (2,7,8). Alternative Cofactors and Substrates: Mn2+, 0.1mM optimal (Mn2+ at 0.1mM results in activity 4X lower than Mg2+ at 6mM) (6); biotin-11-dUTP (15). Inhibitors: T4 DNA Polymerase is reported to be inhibited by -SH blocking reagents (7). T4 DNA Polymerase is sensitive to the concentration of NaCl (20mM, 90% activity; 200mM, 5% activity), KCl (20mM, 75% activity; 200mM, 5% activity), Tris (67mM, 100% activity; 200mM, 50% activity), sodium acetate (50mM, 130% activity; 200mM 40% activity), potassium phosphate (20mM, 100% activity; 100mM, 20% activity) and ammonium sulfate (20mM, 100% activity; 100mM, 20% activity)*. The presence of 20% glycerol results in 40% reduction in activity*. The presence of 1% Tween® 20 or Triton® X-100 results in a 5% decrease in activity*. T4 DNA Polymerase is sensitive to inhibition by various nucleotide/nucleoside analogs (9–12) and aphidicolin (9–12). Pyrophosphate analogs phosphoroacetate (PAA) and phosphoroformate (PFA) are weak inhibitors of T4 DNA Polymerase (11). Figures 2, 3 and 4 provide information on the effects of various restriction enzyme buffers, salt concentrations and temperatures, respectively, on T4 DNA Polymerase. Ki : For aphidicolin and nucleotides BuAdATP and BuPdGTP, refer to references 9 and 10. Temperature Stability: Optimal temperature is 37˚C (25% polymerase activity at 25˚C and 45˚C). Some sources recommend use of the enzyme at 11–12˚C in 3′-end labeling applications to maximize the ratio of polymerase activity to exonuclease activity (13,14). Inactivation: 75˚C, 10 minutes. Error Rate: 1 x 10 –7 (1,16). Processivity: 100–800 nucleotides per binding event. Processivity is templateand enzyme-concentration dependent (1). Polymerization Rate: Approximately 250 nucleotides per second (17). Strand Displacement Activity: No. Genetic Locus: T4 gene 43. * Data was generated at Promega, using unit definition reaction conditions. 25˚C 45˚C 65˚C 0 20 40 60 80 100 120 140 160 Activity (percent control) Temperature Tested (˚C) T4 DNA Polymerase DNA Polymerase I (Large) Klenow Fragment DNA Polymerase I (Large) Klenow Fragment, Exonuclease Minus Figure 4. The influence of various temperatures on DNA polymerase activity. Control activity was determined for each polymerase, using its standard reaction conditions, buffer and temperature, specified for Unit Definition. NaCl KCl 0 20 40 60 80 100 120 140 160 Salt Concentration Resulting in I50 (mM) Salt Tested T4 DNA Polymerase DNA Polymerase I (Large) Klenow Fragment DNA Polymerase I (Large) Klenow Fragment, Exonuclease Minus Figure 3. The concentrations of NaCl and KCl that result in 50% inhibition (I50) of DNA polymerase activity. Activity of each polymerase at these salt concentrations was compared to its activity at reaction conditions specified for Unit Definition. ONE 7 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com ENZYME RESOURCE GUIDE POLYMERASES References 1. Lesley, S.A. et al. (1995) Promega Notes 54, 26. 2. Young, M.C. et al. (1992) Biochemistry 31, 8675. 3. Protocols and Applications Guide, Third Edition (1996) Promega Corporation. 4. Gillin, F.D. and Nossal, N.G. (1975) Biochem. Biophys. Res. Comm. 64, 457. 5. T4 DNA Polymerase Technical Bulletin #TB524, Promega Corporation. 6. Lehman, R. (1981) In: The Enzymes, 3rd Edition, Boyer, P.D., ed., Academic Press, San Diego, California. 7. Kornberg, A. and Baker, T. (1992) In: DNA Replication, W.H. Freeman and Company, San Francisco, California. 8. Mace, D.C. and Alberts, B.M. (1984) J. Mol. Biol. 177, 313. 9. Khan, N.N. et al. (1994) Nucl. Acids Res. 22, 232. 10. Bernad, A. et al. (1987) EMBO J. 6, 4219. 11. Hentosh, P. et al. (1991) Biochemistry 30, 547. 12. Brown, N.C. and Wright, G.E. (1995) Meth. Enzymol. 262, 202. 13. Ausubel, F.M. et al. (1988) In: Current Protocols in Molecular Biology, John Wiley and Sons, New York. 14. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 15. McInnes, J.L. and Symons, R.H. (1990) In: Nucleic Acid Probes, Symons, R.H., ed., CRC Press, Boca Raton, Florida. 16. Kunkel, T.A. et al. (1984) J. Biol. Chem. 259, 1539. 17. Mace, D.C. and Alberts, B.M. (1984) J. Mol. Biol. 177, 295. 18. Spicer, E.K. et al. (1988) J. Biol. Chem. 263, 7478. Promega Product Information Source: Purified from an E. coli strain expressing a recombinant clone. Molecular Weight: T4 DNA Polymerase is a monomeric protein with a calculated molecular weight of 103kDa (18). Molecular weights of 112–114kDa have been reported (13,14). Storage Conditions: Store at –20˚C. Avoid exposure to multiple freeze/thaw cycles. T4 DNA Polymerase is supplied in storage buffer containing: 200mM potassium phosphate (pH 6.5), 2mM DTT and 50% glycerol. Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 55nmol of dTTP into acidprecipitable material in 30 minutes at 39˚C using poly(dA):oligo(dT) as a substrate. See the unit concentration on the Product Information Label. One unit as defined by these conditions is equivalent to one unit under the previous unit definition for this product. Purity: Purity is >95% as determined by SDSpolyacrylamide gel electrophoresis with Coomassie® blue staining. Contaminant Assays Endonuclease Assay: To test for endonuclease activity, 1µg of Type I supercoiled plasmid DNA is incubated with 40 units of T4 DNA Polymerase for one hour at 37˚C in 1X restriction enzyme Buffer D. Following incubation, the DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting. Single-Strand Endonuclease Assay: There must be no endonuclease activity detected when 1µg of M13mp18 DNA is incubated with 40 units of T4 DNA Polymerase for 1 hour at 37˚C. Table 2. The Effect of Promega Restriction Enzyme Core Buffers on T4 DNA Polymerase Activity (see also Figure 2). Activity Buffer (% Control) A ≥ 50% B ≥ 50% C ≥ 50% D < 50% E ≥ 50% G ≥ 50% H ≥ 30% Control activity was determined for T4 DNA Polymerase using reaction conditions specified for Unit Definition. ENZYME RESOURCE GUIDE Notes PROMEGA 8 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com ONE 19 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com ENZYME RESOURCE GUIDE References 1. Butler, E.T. and Chamberlin, M.J. (1982) J. Biol. Chem. 257, 5772. 2. Melton D.A. et al. (1984) Nucl. Acids Res. 12, 7035. 3. Chamberlin, M. and Ryan, T. (1982) In: The Enzymes, Vol XV, Boyer, P.D., ed., Academic Press, New York. 4. Dunn, J.J. and Studier, F.W. (1983) J. Mol. Biol. 166, 477. 5. Schenborn, E.T. and Mierendorf, R.C. (1985) Nucl. Acids Res. 13, 6223. 6. Schenborn, E.T. (1995) In: Methods in Molecular Biology, Tymms, M.J., ed., Humana Press, Totowa, New Jersey. 7. Krieg, P.A. and Melton, D. (1984) Nucl. Acids Res. 12, 7057. POLYMERASES RNA Polymerases The RNA polymerases of bacteriophages T7, T3 and SP6 are single-subunit DNAdependent RNA polymerases that exhibit extremely high specificity for their respective promoter sequences (1,2). For example, T3 and T7 are closely related bacteriophages, but T3 RNA Polymerase will not utilize a T7 promoter, nor will T7 RNA Polymerase recognize a T3 promoter (3). Of the three commonly used enzymes within this group, T7 RNA Polymerase has been the most extensively studied. T7 RNA Polymerase transcribes the class II and III T7 phage genes, so named for the time of their expression during the phage growth cycle. Class III promoters appear stronger in vitro and in vivo than class II promoters (4) and are the promoters commonly used for in vitro transcription vectors. Table 9 provides the phage promoter and leader sequences present in some commonly used in vitro transcription vectors. The bacteriophage RNA polymerases require a DNA template with the appropriate phage promoter, but synthesis does not require a primer as with the DNA polymerases. In vitro transcripts can be produced from a circularized plasmid template, but messages of varying length will result. Discrete RNA transcripts without vector sequences can be produced in two ways. First, transcription can be terminated by the presence of the specific phage RNA polymerase terminator sequence, although some read-through may occur (Figure 1e). The other more common method is to generate run-off transcripts from a template linearized by restriction enzyme digestion (Figure 1f). One complication can arise using the latter procedure if a restriction enzyme that generates 3′-overhangs is used (5). In this instance, transcripts complementary to the opposite strand and vector sequences will be produced, as the polymerase wraps around and begins transcription of the complementary strand. To avoid this, the 3′-overhangs should be converted to blunt ends using any of a number of standard methods (6). Because of the polymerases’ promoter specificity, only DNA downstream of the specific phage promoter sequence will be transcribed. Highly specific, single-stranded RNA produced in vitro by this method can be used in a wide variety of molecular biology applications. If a radiolabeled or tagged (e.g., biotinylated) rNTP is present, RNA probes can be produced. These probes provide hybrids with a higher melting temperature (RNA:RNA > RNA:DNA > DNA:DNA) that may be used in blotting and in situ hybridization procedures. By adjusting reaction conditions, large amounts of RNA can be made, making this a useful means to produce substrates for translation, processing and antisense RNA studies or to study ribozymes, tRNAs or rRNAs. Cap analogs can be easily added to the in vitro transcripts, which may enhance translation of these messages in vitro (7). Table 9. Commonly Used Bacteriophage Promoter and Leader Sequences. The actual start site of transcription is indicated by ^ and the 6-base leader following the ^ will appear in the transcript. Changes to the leader sequence can also impact in vivo and in vitro transcriptional levels. SP6 Promoter/Leader Sequence Vectors 5′-ATT TAGGT GACAC TATA^G AATAC-3′ pGEM®-Zf Vectors(i); pGEM®-3Z and 4Z Vectors(i); pSP64, pSP65, pSP64 Poly(A), pSP70, pSP71 and pSP72 Vectors(i); pGEMEX®-1 and -2 Vectors(i,j,z); pALTER® Vectors; LambdaGEM® Vectors(i) T7 Promoter/Leader Sequence Vectors 5′-TAA TACGA CTCAC TATA^G GGCGA-3′ pGEM®-Zf Vectors; pGEM®-3Z and 4Z Vectors; pALTER® 5′-TAA TACGA CTCAC TATA^G GGAGA-3′ Vectors; pET Vectors(i,j,z); RiboMAX™(a,k,l) and TNT® Luciferase Control DNA(l); pGEMEX®-1 Vector; pGEM®-2 Vector; LambdaGEM® Vectors T3 Promoter/Leader Sequence Vectors 5′-TTA TTAAC CCTCA CTAAA^GGGAAG-3′ pGEMEX®-1 and -2 Vectors; 5′-AAA TTAAC CCTCA CTAAA^GGGAAT-3′ RiboMAX™ and TNT® Luciferase Control DNA PROMEGA ENZYME RESOURCE GUIDE 20 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com T7 RNA Polymerase E.C. 2.7.7.6 Description T7 RNA Polymerase (Cat.# P2075, P2077, P4074) is a DNA-dependent RNA polymerase with an extremely high specificity for its cognate promoter sequence. The enzyme catalyzes the repetitive addition of nucleotide triphosphates on a ss- or dsDNA template. The enzyme does not require a primer to initiate polymerization, but does require that the promoter sequence be double-stranded (1,2). T7 RNA Polymerase is used for the in vitro synthesis of labeled RNA for use as hybridization probes and unlabeled RNA for in vivo or in vitro translation (3–5). Applications •Synthesis of RNA transcripts for hybridization probes (3,4). •Synthesis of large amounts of nonlabeled RNA (3,5). •Synthesis of in vitro capped RNA transcripts (3–5). T7 RNA Polymerase is a component of the following Promega systems: •Riboprobe® System-T7(a) (Cat.# P1440) •Riboprobe® Combination System-SP6/T7 RNA Polymerase(a) (Cat.# P1460) •Riboprobe® Combination System-T3/T7 RNA Polymerase(a) (Cat.# P1450) •RiboMAX™ Large Scale RNA Production System-T7(a,k,l) (Cat.# P1300) •TNT® T7 Coupled Wheat Germ Extract System(l,m,n) (Cat.# L4140) •TNT® T7/SP6 Coupled Wheat Germ Extract System(l,m,n) (Cat.# L5030) •TNT® T7/T3 Coupled Wheat Germ Extract System(l,m,n) (Cat.# L5040) Enzyme Properties Requirements: Mg2+; thiol-reducing agent such as DTT (6). Optimal Substrate: Both ss- and dsDNA can be used; however, the promoter sequence must be double-stranded (1,2). T7 DNA Polymerase can use both supercoiled and linear dsDNA as a template. Extraneous transcripts complementary to the opposite strand are generated from dsDNA templates with 3′-overhanging ends. Use linear templates with blunt ends or 5′-overhangs (3,7). Optimal pH: 7.7–8.3 (6). Alternative Cofactors and Substrates: T7 RNA Polymerase can incorporate biotinylated (8) and digoxigenin-modified nucleotides (9). Inhibitors: T7 RNA Polymerase is inhibited by actinomycin D, heparin, poly r(U) and high salt concentrations (Figure 10; 10). The presence of 2.5% PEG results in a 20% reduction in transcription activity (11). The presence of 5% ethanol results in a 10% reduction in transcription activity (11). T7 RNA Polymerase has variable activity in Promega Restriction Enzyme Core Buffers (Figure 9). Km: ATP, 47µM; CTP, 81µM; GTP, 160µM; UTP, 60µM (6). Temperature Stability: Optimal temperature is 37˚C. In some cases, transcription at temperatures lower than 37˚C may result in a higher proportion of full-length transcripts produced (12). T7 RNA Polymerase is inactivated at temperatures greater than 50˚C (11). Inactivation: Phenol:chloroform extraction or 70˚C, 10 minutes (11). Polymerization Rate: 200–300 nucleotides per second at 37˚C (13). Strand Displacement Activity: Yes. Genetic Locus: T7 gene 1. A B CD E GH 0 20 40 60 80 100 120 Enzyme Activity (percent control) Restriction Enzyme Buffer T3 RNA Polymerase T7 RNA Polymerase SP6 RNA Polymerase NaCl KCl 0 50 100 150 200 250 Salt Concentrations Resulting in I50 (mM) Salt Tested T3 RNA Polymerase T7 RNA Polymerase SP6 RNA Polymerase Figure 10. The concentrations of NaCl and KCl that result in 50% inhibition (I50 ) of RNA Polymerase activity. Activity of each polymerase at these salt concentrations was compared to its activity at standard reaction conditions, specified for Unit Definition. Figure 9. RNA Polymerase activity in Promega Restriction Enzyme Core Buffers (see Table 11). Control activity was determined for each polymerase, using its standard reaction conditions, including buffer composition and temperature, as specified for Unit Definition. ONE 21 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com ENZYME RESOURCE GUIDE POLYMERASES Promega Product Information Source: Purified from an E. coli strain expressing a recombinant clone. Molecular Weight: T7 RNA Polymerase is a monomeric protein with a calculated molecular weight of 98kDa (14). The protein has an apparent molecular weight of 107–110kDa on SDS-polyacrylamide gels (6,13). Storage Conditions: Store at –20˚C. T7 RNA Polymerase is supplied in storage buffer containing 20mM potassium phosphate buffer (pH 7.7), 1mM EDTA, 10mM DTT, 0.1M NaCl, 0.1% Triton® X-100 and 50% (v/v) glycerol. Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 5nmol of CTP into acidinsoluble product in one hour at 37˚C (15). For unit definition the reaction conditions are: 40mM Tris-HCl (pH 7.9), 10mM NaCl, 6mM MgCl2, 10mM DTT, 2mM spermidine, 0.05% Tween®20, 0.5mM each of ATP, GTP, CTP and UTP, 0.5µCi [3H]CTP and 2µg of supercoiled pGEM®-5Zf(+) Vector DNA(i) (Cat.# P2241). The final volume is 100µl. Purity: The purity is >90% as judged by SDS-polyacrylamide gel electrophoresis with Coomassie® blue staining. Activity Assays RNA Synthesis Assay: T7 RNA Polymerase is tested for RNA synthesis using the same conditions as for Unit Definition except unlabeled CTP is limited to 12µM. pGEM® Express Positive Control DNA(i) (20ng/µl; Cat.# P2561) is used as template. Separate reactions are performed using 1, 2, 5, 10 and 20 units of enzyme for 1 hour at 37˚C. Minimum passing specification is >65% incorporation of [3H]CTP using 20 units of enzyme. Transcription Assay: T7 RNA Polymerase is tested in a transcription assay (20ng/µl) using pGEM® Express Positive Control DNA(i) incubated for 1 hour at 37˚C with 5 or 10 units of enzyme. Transcripts are denatured by heating at 65˚C for 10 minutes in formamide/ formaldehyde buffer and resolved on a 1% agarose gel in TAE buffer. Specification is to obtain intact transcripts of the correct size with no degradation. Contaminant Assay Exonuclease Assay: To test for DNase and RNase activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated with 100 units of T7 RNA Polymerase in transcription optimized 1X Buffer for one hour at 37˚C (Table 12). The release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is <1% release for DNase and RNase activity. References 1. Milligan, J.F. et al. (1987) Nucl. Acids. Res. 15, 8783. 2. Milligan, J.F. and Uhlenbeck, O.C. (1989) Meth. Enzymol. 180, 51. 3. Schenborn, E.T. (1995) In: Methods in Molecular Biology, Vol. 37, Tymms, M.J., ed., Humana Press, Totowa, New Jersey. 4. Riboprobe® in vitro Transcription Systems Technical Manual #TM016, Promega Corporation. 5. RiboMAX ™ Large Scale RNA Production System Technical Bulletin #TB166, Promega Corporation. 6. Chamberlin, M. and Ring, J. (1973) J. Biol. Chem. 248, 2235. 7. Schenborn, E.T. and Mierendorf, R.C. (1985) Nucl. Acids Res. 13, 6223. 8. Langer, P.R., Waldrop, A.A. and Ward, D.C. (1981) Proc. Natl. Acad. Sci. USA 78, 6633. 9. Aigner, S. and Pette, D. (1990) Histochemistry 95, 11. 10.Chamberlin, M. and Ring, J. (1973) J. Biol. Chem. 248, 2245. 11.Promega Notes (1991) 29, 6. 12.Krieg, P.A. (1990) Nucl. Acids. Res. 18, 6463. 13.Chamberlin, M. and Ryan, T. (1982) In: The Enzymes, 3rd Ed., Vol. XV, Part B, Boyer, P.D., ed., Academic Press, New York. 14.Stahl, S.J. and Zinn, K. (1981) J. Mol. Biol. 148, 481. 15.Knoche, K., Stevens, J. and Bandziulis, R. (1997) Promega Notes 61, 2. PROMEGA ENZYME RESOURCE GUIDE 22 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com References 1. Schenborn, E.T. (1995) In: Methods in Molecular Biology, Vol. 37. Tymms, M.J., ed., Humana Press, Totowa, New Jersey. 2. Riboprobe ® in vitro Transcription Systems Technical Manual #TM016, Promega Corporation. 3. RiboMAX ™ Large Scale RNA Production System Technical Bulletin #TB166, Promega Corporation. 4. Langer, P.R., Waldrop, A.A. and Ward, D.C. (1981) Proc. Natl. Acad. Sci. USA 78, 6633. 5. Schenborn, E.T. and Mierendorf, R.C. (1985) Nucl. Acids Res. 13, 6223. 6. Promega Notes (1991) 29, 6. 7. Aigner, S. and Pette, D. (1990) Histochemistry 95, 11. 8. Chamberlin, M. and Ryan, T. (1982) The Enzymes, 3rd Ed., Vol. XV, Part B, Boyer, P.D., ed., Academic Press, New York. 9. McGraw, N.J. et al. (1985) Nucl. Acids Res. 13, 6753. T3 RNA Polymerase E.C. 2.7.7.6 Description T3 RNA Polymerase (Cat.# P2083, P4024) is a DNA-dependent RNA polymerase with an extremely high specificity for its cognate promoter sequence. The enzyme catalyzes the repetitive addition of nucleotide triphosphates on a DNA template. T3 RNA Polymerase does not require a primer to initiate polymerization, but does require that the promoter sequence be double-stranded. T3 RNA Polymerase is used for the in vitro synthesis of labeled RNA for use as hybridization probes and unlabeled RNA for in vivo and in vitro translation (1–3). Applications •Synthesis of RNA transcripts for hybridization probes (1,2). •Synthesis of large amounts of nonlabeled RNA (1,3). •Synthesis of in vitro capped RNA transcripts (1–4). T3 RNA Polymerase is a component of the following Promega systems: •Riboprobe® System-T3(a) (Cat.# P1430) •Riboprobe® Combination System-T3/T7 RNA Polymerase(a) (Cat.# P1450) •RiboMAX™ Large Scale RNA Production System-T3(a,k,l) (Cat.# P1290) •TNT® T3 Coupled Wheat Germ Extract System(l,m,n) (Cat.# L4120) •TNT® T7/T3 Coupled Wheat Germ Extract System(l,m,n) (Cat.# L5040) Enzyme Properties Requirements: Mg2+. Optimal Substrate: T3 DNA Polymerase can use both supercoiled and linear dsDNA as a template. Extraneous transcripts complementary to the opposite strand are generated from dsDNA templates with 3′ overhanging ends. Use linear templates with blunt ends or 5′-overhangs (5,6). Alternative Substrates: T3 RNA Polymerase can incorporate biotinylated and digoxigenin-modified nucleotides (4,7). Inhibitors: T3 RNA Polymerase is inhibited by salts (Figure 10). 2.5% PEG results in a 40% reduction in transcription activity (6). The presence of 5% ethanol does not result in reduction in transcription activity (6). T3 RNA Polymerase has variable activity in Promega Restriction Enzyme Core Buffers (Figure 9). Temperature Stability: Optimal temperature is 37˚C. T3 RNA Polymerase is inactivated at temperatures greater than 50˚C (1). Inactivation: Phenol:chloroform extraction or 70˚C, 10 minutes (6). Polymerization Rate: 200–300 nucleotides per second (8) at 37˚C. Strand Displacement Activity: Yes. Genetic Locus: T3 gene 1. Promega Product Information Source: Purified from an E. coli strain expressing a recombinant clone. Molecular Weight: T3 RNA Polymerase is a monomeric protein with a calculated molecular weight of approximately 98.8kDa (9). Storage Conditions: Store at –20˚C. T3 RNA Polymerase is supplied in storage buffer containing 20mM potassium phosphate buffer (pH 7.7), 1mM EDTA, 10mM DTT, 0.1M NaCl, 0.1% Triton® X-100 and 50% (v/v) glycerol. Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 5nmol of CTP into acid-insoluble product in one hour at 37˚C. For unit definition the reaction conditions are: 40mM Tris-HCl (pH 7.9), 10mM NaCl, 6mM MgCl2, 10mM DTT, 2mM spermidine, 0.05% Tween® 20, 0.5mM each of ATP, GTP, CTP and UTP, 0.5µCi [3H]CTP and 2µg of supercoiled pSP6/T3 vector DNA. The final volume is 100µl. Purity:T3 RNA Polymerase is judged to be greater than 90% pure as indicated by SDS-polyacrylamide gel electrophoresis and Coomassie® blue staining. Activity Assays RNA Synthesis Assay: T3 RNA Polymerase is tested for RNA synthesis using the same conditions as for unit definition (above) except unlabeled CTP is limited to 12µM. pGEM® Express Positive Control DNA(i) (20ng/µl; Cat.# P2561) is used as template. Separate reactions are performed using 1, 2, 5, 10 and 20 units of enzyme for 1 hour at 37˚C. Minimum passing specification is ≥65% incorporation of [3H]CTP, using 20 units of enzyme. Transcription Assay: T3 RNA Polymerase is tested in a transcription assay using pGEM® Express Positive Control DNA (20ng/µl) incubated for 1 hour at 37˚C with 5 or 10 units of enzyme. Transcripts are denatured by heating at 65˚C for 10 minutes in formamide/ formaldehyde buffer and resolved in a 1% agarose gel in TAE buffer. Specification is to obtain intact transcripts of the correct size with no degradation. Contaminant Assay Exonuclease Assay: To test for DNase and RNase activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated with 100 units of T3 RNA Polymerase in Transcription Optimized 1X Buffer for one hour at 37˚C (Table 12). The release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is <1% release for DNase and RNase activity. ONE 23 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com SP6 RNA Polymerase E.C. 2.7.7.6 Description SP6 RNA Polymerase (Cat.# P1081, P1085, P4084) is a DNA-dependent RNA polymerase with an extremely high specificity for its cognate promoter sequence. The enzyme catalyzes the repetitive addition of nucleotide triphosphates on a ss- or dsDNA template. The enzyme does not require a primer to initiate polymerization but does require the promoter sequence to be double-stranded (1). SP6 RNA Polymerase is used for the in vitro synthesis of labeled RNA for use as hybridization probes and unlabeled RNA for in vivo or in vitro translation (2–4). Applications •Synthesis of RNA transcripts for hybridization probes (2,3). •Synthesis of large amounts of nonlabeled RNA (2,4). •Synthesis of in vitro capped RNA transcripts (2,4). SP6 RNA Polymerase is a component of the following Promega systems: •Riboprobe® System-SP6(a) (Cat.# P1420) •Riboprobe® Combination System-SP6/T7 RNA Polymerase(a) (Cat.# P1460) •RiboMAX™ Large Scale RNA Production System-SP6(a,k,l) (Cat.# P1280) •TNT® SP6 Coupled Wheat Germ Extract System(l,m,n) (Cat.# L4130) •TNT® T7/SP6 Coupled Wheat Germ Extract System(l,m,n) (Cat.# L5030) Enzyme Properties Requirements: Mg2+; thiol-reducing reagents, such as DTT (5). Optimal Substrate: Transcription is most efficient using dsDNA, although ssDNA can be used, provided that the promoter sequence is double-stranded (2). SP6 RNA Polymerase can use both supercoiled and linear dsDNA as a template. Extraneous transcripts complementary to the opposite strand are generated from dsDNA templates with 3′ overhanging ends. Use linear templates with blunt ends or 5′-overhangs (6). Optimal pH: 7.5 in Tris-HCl buffer; optimal range is pH 7–8.5 (5). Km: ATP, 67µM; CTP, 30µM; GTP, 50µM; UTP, 31µM (5). Inhibitors: SP6 RNA Polymerase is inhibited by salts (Figure 10); activity decreases up to 50% in 20mM (NH4)2SO4 (5). The presence of 2.5% PEG results in a 40% reduction in transcription activity (7). The presence of 5% ethanol results in no reduction in transcriptional activity (7). SP6 RNA Polymerase has variable activity in Promega Restriction Enzyme Core Buffers (Figure 9). Temperature Stability: Optimal temperature is 37˚C. SP6 RNA Polymerase is inactivated at temperatures greater than 50˚C (7). Inactivation: Phenol:chloroform extraction or heat-inactivation at 70˚C for 10 minutes (7). Strand Displacement Activity: Yes. Promega Product Information Source: Purified from an E. coli strain expressing a recombinant clone. Molecular Weight: SP6 RNA polymerase is a monomeric protein with a calculated molecular weight of 98.6kDa (8). Storage Conditions: Store at –20˚C. SP6 RNA Polymerase is supplied in storage buffer containing 20mM potassium phosphate buffer (pH 7.7 at 25˚C), 1mM EDTA, 10mM DTT, 0.1M NaCl, 0.1% Triton® X-100 and 50% (v/v) glycerol. Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 5nmol of CTP into acidinsoluble product in one hour at 37˚C. For unit definition the reaction conditions are: 40mM Tris-HCl (pH 7.9), 10mM NaCl, 6mM MgCl2, 10mM DTT, 2mM spermidine, 0.05% Tween®- 20, 0.5mM each of ATP, GTP, CTP and UTP, 0.5µCi [3H]CTP and 2µg of supercoiled pGEM®-5Zf(+) Vector DNA(i) (Cat.# P2241). The final volume is100µl. Purity: The purity is judged to be greater than 90% as indicated by SDS-polyacrylamide gel electrophoresis and Coomassie® blue staining. Activity Assays RNA Synthesis Assay: SP6 RNA Polymerase is tested for RNA synthesis using the same conditions as for Unit Definition (above), except that the unlabeled CTP is limited to 12µM. pGEM® Express Positive Control DNA(i) (20ng/µl; Cat.# P2561) is used as the template; the reaction volume is 50µl. Separate reactions are performed using 1, 2, 5, 10 and 20 units of enzyme for 1 hour at 37˚C. Minimum passing specification is >65% incorporation of [3H]CTP using 20 units of enzyme. Transcription Assay: SP6 RNA Polymerase is tested in a transcription assay using pGEM® Express Positive Control DNA incubated for 1 hour at 37˚C with 5 or 10 units of enzyme. Transcripts are denatured by heating at 65˚C for 10 minutes in formamide/formaldehyde buffer and resolved in a 1% agarose gel in TAE buffer. Specification is to obtain intact transcripts of the correct size with no degradation. Contaminant Assay Exonuclease Assay: To test for DNase and RNase activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated with 100 units of SP6 RNA Polymerase in transcription optimized 1X Buffer for one hour at 37˚C (Table 12). The release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is <1% release for DNase and RNase activity. ENZYME RESOURCE GUIDE References 1. Stump, W.T. and Hall, K.B. (1993) Nucl. Acids Res. 21, 5480. 2. Schenborn, E.T. (1995) In: Methods in Molecular Biology, Vol. 37. Tymms, M.J., ed., Humana Press, Totowa, New Jersey. 3.Riboprobe® in vitro Transcription Systems Technical Manual #TM016, Promega Corporation. 4.RiboMAX™ Large Scale RNA Production System Technical Bulletin #TB166, Promega Corporation. 5. Butler, E.T. and Chamberlin, M.J. (1982) J. Biol. Chem. 257, 5772. 6. Schenborn, E.T. and Mierendorf, R.C. (1985) Nucl. Acids Res. 13, 6223. 7. Promega Notes (1991) 29, 6. 8. Kotani, H. et al. (1987) Nucl. Acids Res. 15, 2653. POLYMERASES ENZYME RESOURCE GUIDE Notes PROMEGA 24 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com ONE 25 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com Reverse Transcriptases The discovery of reverse transcriptases, or RNA-dependent DNA polymerases, and their role in retrovirus infection (1,2) altered molecular biology’s central dogma of DNA→RNA→protein. These findings revealed that reverse transcriptases, which are found in the virion, catalyze the synthesis of a proviral DNA from the virion’s RNA genome at the onset of the infection process. The primary in vitro application of reverse transcriptase is in the production of cDNA libraries in which the enzyme is used to copy RNA into the first strand of the cDNA product to be cloned. Similarly, reverse transcriptases are used in the first step of two-enzyme reverse transcriptase-polymerase chain reaction (RT-PCR) systems to generate the DNA template required for thermostable DNA polymerases. They also are used to map the 5′-ends of mRNAs in RNA sequencing and cDNA probe production. Reverse transcriptases require a primer for synthesis, like other DNA polymerases. For in vitro applications, these primers can be either oligo(dT), which hybridizes to the poly(A)+ tails of eukaryotic mRNAs; random hexamers, which prime the synthesis from internal sites of the RNA template; or a sequence-specific primer, which hybridizes to a known sequence within the RNA template (3). Polymerization from a primer then proceeds as for DNA-dependent DNA polymerases (Figure 1g and 1h). The two commonly used reverse transcriptases, Avian Myeloblastosis Virus Reverse Transcriptase (AMV RT) and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) perform the same reaction, but at different optimum temperatures (AMV, 42˚C; MMLV, 37˚C). AMV RT is the enzyme of choice for templates with high degrees of secondary structure, as more structure can be melted at 42˚C than at 37˚C. Some reverse transcriptases also possess intrinsic 3′- and/or 5′-exoribonuclease (RNase H) activity, similar to the exonuclease activities associated with some DNA-dependent DNA polymerases. These activities are generally used to degrade the RNA template after the first strand of a cDNA is produced. Absence of the 5′-exoribonuclease (RNase H) activity may aid in the production of longer cDNAs (4). Some DNA-dependent DNA polymerases also possess a reverse transcriptase activity, which can be favored under certain conditions. For example, the thermostable DNAdependent DNA Polymerase, Tth DNA Polymerase(d,e), exhibits reverse transcriptase activity when Mn2+ is substituted for Mg2+ in a reaction. ENZYME RESOURCE GUIDE References 1. Baltimore, D. (1970) Nature 226, 1209. 2. Temin, H. and Mizutani, S. (1970) Nature 226, 1121. 3. Protocols and Applications Guide, Third Edition (1996), Promega Corporation. 4. Berger, S.L. et al. (1983) Biochemistry 22, 2365. POLYMERASES ENZYME RESOURCE GUIDE AMV Reverse Transcriptase E.C. 2.7.7.49 Description AMV Reverse Transcriptase (Cat.# M5101, M5108, M9004) catalyzes the polymerization of DNA using template DNA, RNA or RNA:DNA hybrids (1,2). AMV Reverse Transcriptase is the preferred reverse transcriptase for templates with high secondary structure, due to its higher reaction temperature. AMV Reverse Transcriptase is used in a wide variety of applications including first- and secondstrand DNA synthesis (2,3), sequencing of RNA (4,5) and DNA (6,7), RT-PCR(g) and 5′-rapid amplification of cDNA ends (RACE) (8). The enzyme can also be used for primer extension (9), filling in 5′-overhangs (10) and 3′ end-labeling (11). Although the high optimal temperature makes it the enzyme of choice for cDNA synthesis using templates with complex secondary structure, its relatively high RNase H activity limits its usefulness for generation of long cDNAs (>5kb). For these templates M-MLV Reverse Transcriptase may be a better choice. Applications •First-strand/second-strand synthesis of cDNA (2,3). •RT-PCR. •RNA sequencing (4,5). •DNA sequencing (6,7). •Filling in 5′-overhangs where Klenow is not satisfactory (10). •5′-RACE (rapid amplification of cDNA ends) (8). •Primer extension (9). •3′-end labeling (11). •Studies on secondary structure of RNA (12). AMV Reverse Transcriptase is available in the following Promega systems: •Access RT-PCR System(g) (Cat.# A1250, A1260, A1280) •Universal RiboClone® cDNA Synthesis System(a) (Cat.# C4360) •Reverse Transcription System(a) (Cat.# A3500) Enzyme Properties Requirements: A DNA primer is required for efficient reverse transcription of RNA. Primers of greater than 8 nucleotides are most effective. Divalent cations such as Mg2+ or Mn2+ at 6–10mM are also required (13). Mn2+ has been reported to increase the DNA endonuclease activity of AMV Reverse Transcriptase. Cofactors: Sodium pyrophosphate and spermidine help drive the DNA polymerase activity forward and denature RNA secondary structure, respectively (11). Optimal Substrate: ssDNA or RNA. Fifteen units of enzyme per microgram of RNA is commonly used for first-strand synthesis (13). Optimal pH: 8.3 in Tris-HCl buffer. Inhibitors: DNA-dependent DNA polymerase activity is strongly inhibited by actinomycin D at 50µg/ml final concentration (9,14). Nucleic acids rRNA and tRNA (15) may also inhibit the enzyme. High amounts of secondary structure in the RNA being transcribed can inhibit chain elongation. Glycerol concentrations >10% may inhibit up to 50% of activity. Ribonucleoside-vanadyl complexes at 2mM or above can inhibit AMV Reverse Transcriptase activity, unless all four dNTPs exceed approximately 1mM. N-ethyl maleimide inactivates AMV RT(10). Temperature Stability: 37–58˚C. Typical working conditions are at 42˚C, but for RT-PCR applications, temperatures as high as 58˚C will yield acceptable results (16). Inactivation: Addition of 50mM EDTA (pH 8.0) heating to 75˚C for 15 minutes. Alternatively, 100˚C for 5 minutes followed by a 5-minute incubation on ice. If analyzing reverse transcription on native gels, do not heat as this may cause RNA/cDNA hybrids to form, making interpretation difficult. Error Rate: Due to the lack of proofreading 3′→5′ exonuclease activity, error rates for all reverse transcriptases are high. The average error rate for AMV RT is 4.9×10–4, based on misincorporation studies (17). In addition, AMV RT will only resume faithful incorporation 25% of the time after a misinsertion. Promega Product Information Source: Purified from Avian myeloblastosis virus particles. Molecular Weight: AMV Reverse Transcriptase is a 170kDa heterodimer with an α-subunit of 65kDa and a β-subunit of 94kDa (18). The smaller subunit is a proteolytic product of the larger subunit (1,2). Typical Working Conditions: 50mM Tris-HCl (pH 8.3 at 25˚C), 50mM KCl, 5mM MgCl2, 5mM DTT, 40µM each dNTP, 0.1mg/ml BSA. Storage Conditions: Store at –20˚C. Avoid exposure to frequent temperature changes. AMV Reverse Transcriptase is supplied in storage buffer containing 200mM potassium phosphate (pH 7.2), 0.2% Triton® X-100, 2mM DTT and 50% (v/v) glycerol. PROMEGA 26 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com ONE 27 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com Unit Definition: One unit is defined as the amount of enzyme required to catalyze the transfer of 1nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37˚C. For unit definition the reaction conditions are: 50mM Tris-HCl (pH 8.3), 40mM KCl, 8.75mM MgCl2, 10mM DTT, 0.1mg/ml BSA, 1mM [3H]-labeled dTTP, 0.25mM poly(A)400 and 0.25mM oligo(dT)50. Purity: The enzyme is greater than 80% pure, as judged by SDS-polyacrylamide gel electrophoresis with Coomassie® blue staining. Activity Assay Functional Assay: First-strand cDNA of a 1.2kb Control RNA (Cat.# C1381) is synthesized using 15 units of AMV Reverse Transcriptase, 1µg of template, 0.5µg of oligo(dT)15 primer and 1mM each dNTP for 60 minutes at 42˚C. The minimum specification is the production of >120ng of first-strand cDNA. Full-length cDNA must be observed following gel electrophoresis and autoradiography. Contaminant Assays Endonuclease Assay: To test for endonuclease activity, 0.5µg of Type I supercoiled plasmid DNA is incubated with 25 units of AMV Reverse Transcriptase in 50mM Tris (pH 8.3), 40mM KCl, 7mM MgCl2 and 10mM DTT for one hour at 37˚C. Following incubation, the DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of nicking or cutting. Exonuclease Assay: To test for DNase and RNase activity, 50ng of radiolabled DNA or radiolabeled RNA is incubated with 25 units of AMV Reverse Transcriptase in 50mM Tris (pH 8.3), 40mM KCl, 7mM MgCl2 and 10mM DTT for one hour at 37˚C. The release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is <1% release for DNase and 3% release for RNase. ENZYME RESOURCE GUIDE References 1.Kacian, D.L. (1977) Meth. Virol. 6, 143. 2.Houts, G.E. et al. (1979) J.Virol. 29, 517. 3.Gubler, U. and Hoffman, B.J. (1983) Gene 25, 263. 4.Kaartinen, M. et al. (1983) J. Immunol. 130, 937. 5.Fujinaga, K. et al. (1970) Proc. Natl. Acad. Sci. USA 67, 1432. 6.Zagursky, R.J. et al. (1985) Genet. Anal. Tech. 2, 89. 7.Bartlett, J. et al. (1986) BioTechniques, 4, 208. 8.Skinner, T.L., Kerns, R.T. and Bender, P.K. Gene 151, 247. 9.Maniatis, T. et al. (1976) Cell 8 163. 10.Gorecki, M. and Panet, A. (1978) Biochemistry 17, 2438. 11.Oyama, F. et al. (1988) Anal. Biochem. 172, 444. 12.Inoue, T. and Cech, T.R. (1985) Proc. Natl. Acad. Sci. USA 82, 648. 13.AMV Reverse Transcriptase Promega Product Information #9PIM510, Promega Corporation. 14.Sambrook, J. et al. (1989) In: Molecular Cloning: A Laboratory Manual, Second Edition, CSH Press, USA. 15.Yamawa, I. and Caralieri, L.F. (1978) J. Virol. 27, 300. 16.Miller, K. and Storts, D.R. (1995) Promega Notes 53, 2. 17.Ricchetti, M. and Buc, H. (1990) EMBO J. 9, 1583. 18.Grandgenett, D.P. et al. (1973) Proc. Natl. Acad. Sci. USA 70, 230. POLYMERASES ENZYME RESOURCE GUIDE References 1. Roth, M.J., Tanese, N. and Goff, S.P. (1985) J. Biol. Chem. 260, 9326. 2. Verma, I.M. (1974) In: The Enzymes, Boyer, P.D., ed., Academic Press, New York, 87. 3. M-MLV Reverse Transcriptase Technical Bulletin #TB545, Promega Corporation. 4. Gerard, G.F. (1983) In: Enzymes of Nucleic Acid Synthesis and Modification: DNA Enzymes, CRC Press, Boca Raton, Florida, 1. 5. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning, A Laboratory Manual Vol. 1, Second Edition, 5.34. 6. Schaefer, B.C. (1995) Anal. Biochem. 227, 255. 7. Ricchetti, M. and Buc, H. (1990) EMBO J. 9, 1583. 8. Krug, M.S and Berger, S.L. (1987) Meth. Enzymol. 152, 316. M-MLV Reverse Transcriptase (M-MLV RT) E.C. 2.7.7.49 Description M-MLV RT (Cat.# M1701, M1705) is a single polypeptide RNA-dependent DNA polymerase. The enzyme also has DNAdependent DNA polymerase activity at higher enzyme levels (1,2). M-MLV RT is used in a variety of applications including cDNA synthesis, RNA sequencing (3), RT-PCR(g) and 5′-RACE (4). The enzyme can be used for primer extension (3) and filling in 5′-overhangs (2). Its relatively low RNase H activity compared to AMV RT (5) makes M-MLV RT the choice for generation of long cDNAs (>5kb). However, for short templates with complex secondary structure, AMV RT may be a better choice due to its higher optimal temperature. M-MLV RT is less processive than AMV RT; more units of M-MLV RT may be required to generate the same amount of cDNA (6). Applications •cDNA synthesis of mRNA. •RT-PCR. •RACE (rapid amplification cDNA ends) (4). •mRNA 5′-end mapping by primer extension analysis (3). •3′-termini fill-in of dsDNA with 5′-protruding ends (2). •RNA sequencing using ddNTPs (3). Enzyme Properties Requirements: Mg2+ and/or Mn2+. Optimal Substrate: RNA template and DNA primer of 9–15 bases. Optimal pH: 8.0 at 37˚C with Tris-HCl. Km: The Km varies considerably for the individual dNTPs, from 0.5 to 80µM, depending upon the nucleotide and template sequence. A Km of 18µM was determined for all 4 dNTPs combined, using the HIV p25 gag template (7). Alternative Cofactors and Substrates: RNA can also act as a primer, but is much less efficient than DNA (2); 6mM MnCl2 (1); ddNTPs (3). Inhibitors: Inorganic phosphate and pyrophosphate (1): 20mM inorganic phosphate or 0.5mM sodium pyrophosphate results in nearly 50% loss of activity; spermidine (8); 50µg/ml actinomycin D inhibits second-strand synthesis (3). Half-Life: 6.5 minutes at 45˚C, with or without template (2); 15 minutes at 42˚C (1). Inactivation: The addition of 50mM EDTA or heating to 70˚C for 10 minutes will inactivate M-MLV RT. Error Rate: Due to the lack of a proofreading 3′→5′ exonuclease activity, error rates for all reverse transcriptases are high. An average error rate of 1 in 1,000 bases has been shown with Mg2+(6,7). M-MLV will only resume faithful incorporation 50% of the time after a misinsertion (7). Promega Product Information Source: Purified from an E. coli strain expressing a recombinant clone (1). Molecular Weight: 71kDa (1). Typical Working Conditions: 3mM MgCl2 is optimal, while 2–4mM results in >80% activity and >10mM results in <50% activity; 75mM KCl and NaCl is optimal, while 0–125 results in >70% activity (1); 10mM DTT, 0.5–2mM each dNTP; 0.1mg/ml BSA. Storage Conditions: Store at –20˚C. Avoid exposure to frequent temperature changes. M-MLV RT is supplied in storage buffer containing 20mM Tris-HCl (pH 7.5), 200mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01% Nonidet® P-40 and 50% (v/v) glycerol. Unit Definition: One unit is defined as the amount of enzyme required to catalyze the transfer of 1nmol of deoxynucleotide into acid-precipitable material in 10 minutes at 37˚C. For unit definition the reaction conditions are: 0.5mM [3H]dTTP, 0.025mM oligo(dT)50 and 0.25mM poly(A)400 in 50mM Tris-HCl (pH 8.3), 7mM MgCl2, 40mM KCl, 10mM DTT and 0.1mg/ml BSA. Purity: Purity is >90% as judged by SDSpolyacrylamide gel electrophoresis with Coomassie® blue staining. Activity Assay Functional Assay: First-strand cDNAs, of 1.2kb and 7.5kb control RNAs, are synthesized using 200 units of M-MLV RT, 1µg of each template, 0.5µg oligo(dT)15 primer 0.5mM each dNTP and a radiolabeled dCTP at 37˚C for 1 hour in 25µl. The minimum specification is the production of 120ng of first-strand cDNA. In addition, >90% full-length cDNA for the 1.2kb RNA must be observed by gel electrophoresis and autoradiography. Contaminant Assays Endonuclease Assay: To test for endonuclease activity, 1µg of Type I supercoiled plasmid DNA is incubated with 500 units of M-MLV Reverse Transcriptase in 1X reaction buffer for 1 hour at 37˚C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained gel to verify the absence of visible nicking or cutting. Exonuclease Assay: To test for DNase and RNase activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated with 200 units of M-MLV Reverse Transcriptase in 1X reaction buffer for 1 hour at 37˚C (Table 12). The release of radiolabeled nucleotides is monitored by scintillation counting of TCAsoluble material. Minimum passing specification is <1% release for DNase and <3% for RNase. PROMEGA 28 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com ONE 29 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com M-MLV Reverse Transcriptase, RNase H Minus (M-MLV RT, H Minus) E.C. 2.7.7.49 Description M-MLV Reverse Transcriptase, RNase H Minus (Cat.# M5301*), is an RNA-dependent 5′→3′ DNA polymerase that has been genetically altered to remove the associated ribonuclease H activity (1). M-MLV Reverse Transcriptase, RNase H Minus, is used in a wide variety of applications including synthesis of cDNA, RT-PCR(g) and sequencing of RNA (2). The enzyme can also be used for primer extension (2) and filling in 5′-overhangs (3). The absence of RNase H activity of the enzyme makes it the enzyme of choice for generation of long cDNAs (>5kb). However, for shorter templates with complex secondary structure AMV Reverse Transcriptase may be a better choice due to its higher optimal temperature. *Product is only available in certain countries. Applications •cDNA synthesis from mRNA. •RT-PCR. •Primer extension. •mRNA 5′-end mapping by primer extension analysis (2). •3′-termini fill-in of dsDNA with 5′-protruding ends (3). •RNA sequencing using ddNTP (2). Enzyme Properties Requirements: Mg2+ and/or Mn2+. Optimal Substrate: RNA template and DNA primer of 9–15 bases. Optimal pH: 8.0 at 37˚C with Tris-HCl. Km: The Km varies considerably for the individual dNTPs, from 0.5 to 80µM, depending on the nucleotide and template sequence. A Km of 18µM was determined for all 4 dNTPs combined, using the HIV p25 gag template (4). Alternative Cofactors and Substrates: RNA can act as a primer also, but is much less efficient than DNA (3), 6mM MnCl2 (5), ddNTPs (2). Inhibitors: Inorganic phosphate and pyrophosphate (1): 20mM inorganic phosphate or 0.5mM sodium pyrophosphate results in nearly 50% loss of activity; spermidine (3); 50µg/ml actinomycin D inhibits second-strand synthesis (2). Half-Life: 6.5 minutes at 45˚C, with or without template (3); 15 minutes at 42˚C (5). Inactivation: Addition of 50mM EDTA or heating to 70˚C for 10 minutes. Error Rate: Due to the lack of a proofreading 3′→5′ exonuclease activity, error rates for all reverse transcriptases are high. As high as 1 in 500 bases is misincorporated by M-MLV RT, H Minus, in the presence of Mn2+ and high concentrations of dNTPs (6). An average error rate of 1 in 1,000 bases has been shown with Mg2+. M-MLV RT, H Minus, will only resume faithful incorporation 50% of the time after a mismatch (4). Promega Product Information Source: Purified from an E. coli strain expressing a recombinant clone (5). Molecular Weight: 71kDa (5). Typical Working Conditions: 3mM MgCl2 is optimal, while 2–4mM results in >80% activity and >10mM results in <50% activity; 75mM KCl and NaCl is optimal, while 0–125mM results in >70% activity (1); 10mM DTT; 0.5–2mM each dNTP; 0.1mg/ml BSA. Storage Conditions: Store at –20˚C. Avoid exposure to frequent temperature changes. M-MLV RT, H Minus, is supplied in storage buffer containing 20mM Tris-HCl (pH 7.5), 200mM NaCl, 0.1mM EDTA, 1mM DTT, 0.01% Nonidet® P-40 and 50% glycerol. Unit Definition: One unit is defined as the amount of enzyme that incorporates 1nmol of dTTP into acid-insoluble form in 10 minutes at 37˚C. For unit definition the reaction conditions are: 50mM Tris-HCl (pH 8.3), 40mM KCl, 10mM DTT, 7mM MgCl2, 0.1mg/ml BSA, 0.5mM radiolabeled dTTP, 0.25mM poly(A)400 and 0.025mM oligo(dT)50. Purity: Purity is >90% as judged by SDSpolyacrylamide gel electrophoresis with Coomassie® blue staining. Activity Assay Functional Assay: First-strand cDNA of a 1.2kb Control RNA is synthesized using 200 units of M-MLV RT, H Minus. By autoradiography the cDNA product must be observed as a single, full-length band. The minimum passing specification is 12% conversion of RNA into cDNA. The product is labeled with [32P]dCTP. Contaminant Assays Endonuclease Assay: To test for endonuclease activity, 1µg of Type I supercoiled plasmid DNA is incubated with 200 units of M-MLV RT, H Minus, in 1X reaction buffer for 1 hour at 37˚C (Table 12). Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained gel to verify the absence of visible nicking or cutting. Exonuclease Assay: To test for DNase and RNase activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated with 200 units of M-MLV Reverse Transcriptase, H Minus, in 1X reaction buffer for 1 hour at 37˚C (Table 12). The release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is <1% release for DNase and <3% for RNase. RNase H Activity: Two hundred units of enzyme is incubated with polyA:poly(d)T for 1 hour at 37˚C. The release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is <1% release. ENZYME RESOURCE GUIDE POLYMERASES References 1. Tanese, N. and Goff, S. (1988) Proc. Natl. Acad. Sci. USA 85, 1777. 2. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning: A Laboratory Manual, Second Edition. 3. Verma, I.M. (1974) In: The Enzymes, Boyer, P.D., ed., Academic Press, New York. 4. Ricchetti, M. and Buc, H. (1990) EMBO J. 9, 1583. 5. Roth, M.J., Tanese, N. and Goff, S.P. (1985) J. Biol. Chem. 260, 9326. 6. M-MLV Reverse Transcription Product Information Sheet 9P1M170, Promega Corporation. ENZYME RESOURCE GUIDE References 1. Bollum, F.J. (1974) In: The Enzymes, Boyer, P.D., ed., Academic Press, New York, 145. 2. Matsukage, A. et al. (1987) J. Biol. Chem. 262, 8745. 3. Baltimore, D. (1974) Nature 248, 409. 4. Bollum, F.J. (1979) Blood 54, 1203. 5. Bollum, F.J. and Chang, L.M.S. (1986) Adv. Cancer Res. 47, 37. 6. Deng, G., Wu, R. (1983) Meth. Enzymol. 100, 96. 7. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) In: Molecular Cloning, A Laboratory Manual Vol. 1, Second Edition, 5.56. 8. DNA 3´-End Labeling System Technical Bulletin #TB088, Promega Corporation. 9. Barkley, M.D. and Coleman, M.S. (1987) J. Biol. Chem. 262, 9494. 10.Mitra, G. (1996) Promega Notes 57, 10. 11.Bollum, F.J. (1974) In: The Enzymes, Boyer, P.D., ed., Academic Press, New York, 145. 12.Promega Corporation, unpublished results. Terminal Deoxynucleotidyl Transferase E.C. 2.7.7.31 Description Terminal Deoxynucleotidyl Transferase (Cat.# M1871) catalyzes the repetitive addition of mononucleotides from deoxynucleotide triphosphates to the 3′-OH of a DNA initiator, accompanied by the release of inorganic phosphate. Polymerization is not template dependent. ssDNA is preferred as the initiator. The addition of 1mM Co2+ (as CoCl2) in the reaction buffer allows the tailing of any type of 3′-end, with various efficiencies. Of all dsDNA initiators, those with protruding 3′-ends are most efficiently tailed. TdT demonstrates DNA acceptor preference in the following order: ssDNA>dsDNA w/3′-overhangs >dsDNA with 5′-overhangs or blunt ends (6). TdT is used for 3′ end-labeling with labeled or unlabeled nucleotides (2–4). The addition of nucleotides can be limited to a single nucleotide with the use of cordycepin-5′-triphosphate (deoxyadenosine-5′-triphosphate). Tailing DNA with radiolabeled nucleotides will generate probes with higher specific activities than 5′-end-labeling with T4 polynucleotide kinase. However, the addition of multiple nucleotides will change the sequence of the probe and perhaps its specificity. Another common application for TdT is its use in the TUNEL assay (TdT-mediated dUTP Nick End Labeling assay) for apoptosis detection (5,18). Applications •Tailing reactions to add homopolymer tails to DNA vectors and cDNA for cloning (7). •DNA 3′-end labeling, isotope and non-radioactive (8,9). •Apoptosis detection (TUNEL assay (10,22,23). TdT is a component of the following Promega systems: •3′-End Labeling Application Pack (Cat.# M7710) •Apoptosis Detection System, Fluorescein(n) (Cat.# G3250) •DeadEnd™ Colorimetric Apoptosis Detection System (Cat.# G7130) Enzyme Properties Requirements: A divalent metal is required. Mg2+ is preferred for adding purines and cobalt is preferred for adding pyrimidines or rNTPs. Acceptor nucleic acid must be ss- or dsDNA with a free 3′-OH and a minimum of 3 nucleotides in length (7). Cofactor Concentration: 1–2mM CoCl2 (2mM is optimal for dsDNA) or 4–10mM MgCl2 are commonly reported. Optimal Substrate: ssDNA is preferred, but dsDNA with 3′-overhangs, 5′-overhangs or blunt ends will also work with varying efficiency. Optimal Substrate Concentration: The ratio of acceptor DNA to the NTP used will determine, approximately, the number of nucleotides added, i.e., 1pmol 3′-ends to 10pmol nucleotide will add an average of 10 nucleotides per acceptor. For cordycepin triphosphate, the ratio of acceptor to label should be at least 1:1.5 to 1:2. A minimum of 5 units of enzyme should be used for each pmol of acceptor for uniform incorporation (8). Optimal pH: 6.8–7.2 (11,12). PROMEGA 30 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com Terminal deoxynucleotidyl transferase (TdT) is the sole member of this final group of polymerases. Unlike all other polymerases, TdT-directed synthesis does not copy or require a template, and appends nucleotides to the 3′-ends of oligo- and polynucleotide primers in the following manner (see also Figure 1i): Metal2+ ndNTP + d(pX)m d(pX)m(pN)n + nPPi The enzyme is distributive and does not exhibit PPi exchange, pyrophosphorolysis or exonuclease activities. TdT has been used widely for synthesis of homo- and heteropolymers; it can be used to synthesize a wide variety of polymers, since it is very tolerant of nucleotide derivatives (1). TdT is closely related to the repair enzyme DNA Polymerase B (2), but the biological role of TdT remains a mystery. Enzyme activity is only found in pre-T and pre-B lymphocyte nuclei during immunopoiesis, suggesting the enzyme could play a role in generating somatic diversity of immonoglobulin and T-cell receptor genes (3). While its mutator role has not been directly demonstrated, TdT has proven a valuable marker in the diagnosis of leukemia (4,5). ONE 31 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com Km: 100µM for dATP and dGTP; 500µM for dTTP and dCTP; up to 1mM for homopolymer acceptors (11); approximately 1µM for oligonucleotide acceptors (13,16). Stimulator: If using Mg2+ as the primary divalent metal, ZnSO4 in concentrations from 0.33mM to 0.8mM have been shown to improve activity (12,13). Alternative Cofactors and Substrates: Mn2+ (6), [35S]-labeled nucleotide triphosphates (17), ribonucleotide triphosphates, ddNTPs, cordycepin triphosphate, biotinylated nucleotide triphosphates (18), digoxygeninconjugated nucleotide triphosphates (19), fluorescent nucleotide triphosphates and amino-modified triphosphates [NH2-rATP] (20). Inhibitor: Phosphate buffers and high dNTP concentrations, which subsequently produce high pyrophosphate concentrations, should be avoided, as cobalt phosphate and cobalt pyrophosphate are insoluble (14). DTT concentrations above 5mM will also precipitate cobalt. If DNA is ethanolprecipitated prior to TdT tailing, sodium is a better cation than ammonium for enzyme activity. Chloride and iodide are also inhibitory and therefore cacodylate buffers are typically used (15). Tris has the potential to form a complex with Zn2+ and should be avoided (13). Temperature Stability: Avoid temperatures over 40˚C (11). Inactivation: 70˚C for 10 minutes or addition of 20mM EDTA in a reaction containing 1mM CoCl2 and 10 units of TdT (12). Promega Product Information Source: Calf thymus. Molecular Weight: 32,360 daltons with an α-subunit estimated at 8,000 daltons and a β-subunit estimated at 26,500 daltons (21). Storage Conditions: Long-term (use of 1–2 times per month): –70˚C. For daily/weekly use: –20˚C. TdT is supplied in storage buffer containing 50mM potassium phosphate (pH 7.4), 1mM β-mercaptoethanol and 50% glycerol. Unit Definition: One unit of activity catalyzes the transfer of 1 nanomole of dATP to p(dA)50 per hour at 37˚C. For unit definition the reaction conditions are: 40mM cacodylate buffer (pH 6.8 with KOH), containing 8mM MgCl2, 0.33mM ZnSO4, 1mM dATP, 10µM p(dA)50 and 1µCi of [32P]dATP in a 50µl total reaction volume. Reaction Buffer: TdT is provided with a 5X assay buffer consisting of 500mM cacodylate (pH 6.8), 5mM CoCl2 and 0.5mM DTT. Activity Assay Functional Assay: 3′-end labeling is accomplished by incubating 8ng of the positive control oligo(dT)15 primer in 1X assay buffer with 16µCi [α-32P]dATP and 20 units of TdT (final volume 20µl) for 60 minutes at 37˚C. Minimum passing specification is >50% incorporation. Contaminant Assays Endonuclease Assay: To test for endonuclease activity, 1µg of Type I supercoiled plasmid DNA is incubated with 5 units of TdT for one hour at 37˚C in 1X reaction buffer (Table 12) with 10mM MgCl2 added. Following incubation, the DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting. Exonuclease Assay: To test for DNase and RNase activity, 50ng of radiolabeled DNA or radiolabeled RNA is incubated with 25 units of Terminal Deoxynucleotidyl Transferase for one hour at 37˚C in 1X reaction buffer with 10mM MgCl2 added (Table 12). The release of radiolabeled nucleotides is monitored by scintillation counting of TCA-soluble material. Minimum passing specification is <3% for both DNase and RNase. ENZYME RESOURCE GUIDE References (continued) 13.Chang, L.M. and Bollum, F.J. (1990) J. Biol. Chem. 265, 17436. 14.Eschenfeldt, W.H., Puskas, R.S. and Berger, S.L. (1987) Meth. Enzymol. 152, 337. 15.Grosse, F. and Manns, A. (1993) In: Enzymes of Molecular Biology, Burrel, M.M., ed., 16, 95. 16.Kato, K.I. et al. (1967) J. Biol. Chem. 242, 2780. 17.Collins, M.L. and Hunsaker, W.R. (1985) Anal. Biochem. 151, 211. 18.DeBorde, D.C. et al. (1986) Anal. Biochem. 157, 275. 19.Vincent, C. et al. (1982) Nucl. Acids Res. 10, 6787. 20.Protocols and Applications Guide, Third Edition (1996), Promega Corporation, 126. 21.Chang, L.M.S. and Bollum, F.J. (1971) J. Biol. Chem. 246, 909. 22.Hines, M.D. and Allen-Hoffman, B.L. (1996) Promega Notes 59, 30. 23.Apoptosis Detection System, Fluorescein, Technical Bulletin #TB235, Promega Corporation. POLYMERASES PROMEGA ENZYME RESOURCE GUIDE 32 ONE Table 10. Promega’s Quality Acceptance Criteria for Enzymes. CONTAMINANT ACTIVITY CONCENTRATION SDS-PAGE Endonuclease: dsDNase: 50ngRNase: 50ng ENZYME (u/µl) PURITY PCR (g,*) Supercoiled DNA, 1µgRadiolabeled DNA Radiolabeled RNA DNA Polymerase I 5–10 >90% Gel analysis, absence of visible N/A N/A nicking or cutting, 25u/5hr at 37°C DNA Polymerase I 5–10 >90%, Gel analysis, absence of visible N/A N/A Large (Klenow) Fragment no Pol I nicking or cutting, 25u/16hr at 37°C DNA Polymerase I Large 5-10 >90% Gel analysis, absence of visible <2% release, N/A (Klenow) Fragment, Exo Minus nicking or cutting, 25u/16hr at 37˚C 30u/16hr at 37˚C T4 DNA Polymerase1 5–10 >95% Gel analysis, absence of visible N/A N/A nicking or cutting, 40u/1hr at 37°C Taq DNA Polymerase 2 (d) 5 >90% ✓ Gel analysis, absence of visible cutting, N/A N/A 5u/8 hr at 45°C and 8hr at 70°C Tfl DNA Polymerase (d) 5 >90% ✓ Gel analysis, absence of visible <3% release, <3% release, nicking or cutting, 5u/16hr at 74°C 25u/1hr at 74°C 25u/1hr at 48°C Tli DNA Polymerase(d) 3 N/A ✓ Gel analysis, absence of visible N/A N/A nicking or cutting,10u/6hr at 74°C 2–10 N/A Tth DNA Polymerase(d,e) 5 >95% ✓ >90% Form I, 5u/16hr at 70°C <1% release, <3% release, also tested on lambda DNA 5u/1hr at 70°C 5u/1hr at 37°C Pfu DNA Polymerase 3 (d) 2–3 N/A ✓ N/A N/A N/A T7 RNA Polymerase4 10–20 >90% N/A <1% release, <1% release, 80 100u/1hr at 37°C T3 RNA Polymerase4 10–20 >90% N/A <1% release, <1% release, 80 100u/1hr at 37°C SP6 RNA Polymerase4 10–20 >90% N/A <1% release, <1% release, 80 100u/1hr at 37°C AMV Reverse 5–10 >80% Gel analysis, absence of visible <1% release, <3% release, Transcriptase5 20–25 nicking or cutting, 25u/1hr at 37°C 25u/1hr at 37°C 25u/1hr at 37°C M-MLV RT 200 >90% Gel analysis, absence of visible <1% release, <3% release, nicking or cutting, 500u/1hr at 37°C 200u/1hr at 37°C 200u/1hr at 37°C M-MLV Reverse 100–200 >90% Gel analysis, absence of visible <1% release, <3% release, Transcriptase, RNase H Minus nicking or cutting, 200u/1hr at 37°C 200u/1hr at 37°C 200u/1hr at 37°C Terminal Deoxynucleotidyl 15–30 N/A Gel analysis, absence of visible <1% release, <3% release, Transferase nicking or cutting, 5u/1hr at 37°C 25u/1hr at 37°C 25u/1hr at 37°C N/A – Not Applicable Special Assays: Performance Testing 1T4 DNA Polymerase: Free of endonuclease on single-strand circular M13mp18 DNA. 2Taq DNA Polymerase: Free of contaminating exonucleases on lambda/Hind III digested DNA, 5u/8hr at 45˚C and 8hr at 72˚C. 3Pfu DNA Polymerase: Free of contaminating exonucleases on lambda/Hind III digested DNA, 5u/8hr at 45˚C and 8hr at 72˚C. Free of endonucleases on lambda DNA, 12.5u/8hr at 45˚C and 8hr at 72˚C. 4RNA Polymerases: Using a transcription plasmid with the appropriate promoter, an incorporation assay with limiting CTP must result in >65% incorporation of label using 20 units of polymerase. The denatured transcription products are analyzed on agarose gels. 5AMV RT: First-strand cDNA of 1.2kb is synthesized and analyzed by autoradiography. *PCR Satisfaction Guarantee Promega’s PCR Systems, enzymes and reagents are proven in PCR to ensure reliable, high performance results. Your success is important to us. Our products are backed by a worldwide team of Technical Support scientists. Please contact them for applications or technical assistance. If you are not completely satisfied with any Promega PCR product we will send a replacement or refund your account. That’s Our PCR Guarantee! Product must be within expiration date and have been stored and used in accordance with product literature. See Promega Product Insert for specific tests performed. ONE 33 TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com Table 12. Polymerase Reaction Buffer Formulations. Enzyme Dilution Factor Formulation DNA Polymerase I 10X 500mM Tris-HCl (pH 7.2 at 25˚C), 100mM MgSO4, 1mM DTT Klenow Klenow Exo Minus Taq DNA Polymerase1,2 (d) 10X 500mM KCl, 100mM Tris-HCl (pH 9.0 at 25˚C) and 1.0% Tth DNA Polymerase1,3 (d,e) Triton® X-100 Tli DNA Polymerase1 (d) Tfl DNA Polymerase4 (d) 10X 200mM Tris-Acetate (pH 9.0 at 25˚C), 100mM ammonium sulfate, 750mM potassium acetate, 0.5% Tween® 20 Pfu DNA Polymerase (d) 10X 200mM Tris-HCl (pH 8.8 at 25˚C), 100mM KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1% Triton® X-100 and 1mg/ml nuclease-free BSA T7 RNA Polymerase5 5X 200mM Tris-HCl (pH 7.9), 30mM MgCl2, 10mM spermidine T3 RNA Polymerase5 and 50mM NaCl SP6 RNA Polymerase5 AMV Reverse Transcriptase 5X 250mM Tris-HCl (pH 8.3 at 25˚C), 250mM KCl, 50mM MgCl2, 2.5mM spermidine, 50mM DTT M-MLV Reverse Transcriptase 5X 250mM Tris-HCl (pH 8.3 at 25˚C), 375mM KCl, 15mM MgCl2, M-MLV Reverse Transcriptase, 50mM DTT RNase H Minus Terminal Deoxynucleotidyl 5X 500mM cacodylate buffer (pH 6.8), 5mM CoCl2, 0.5mM DTT Transferase (TdT) 1Also provided with 25mM MgCl2. 2Also available with a similar buffer containing 15mM MgCl2. 3Tth DNA Polymerase is also provided with a 10X Reverse Transcription Buffer [100mM Tris-HCl (pH 8.3), 900mM KCl] and a 10X Chelate Buffer [100mM Tris-HCl (pH 8.3), 1mM KCl, 7.5mM EGTA, 0.5% Tween® 20, 50% glycerol]. 4Also provided with 25mM MgSO4. 5Also provided with 100mM DTT. ENZYME RESOURCE GUIDE POLYMERASES Table 11. Composition of Promega Restriction Enzyme Buffers (at 10X) . Promega’s Restriction Enzyme Reaction Buffers are provided as 10X stocks. Buffer pH (at 37°C) Tris-HCl (mM) MgCl2 (mM) NaCl (M) KCl (M) DTT (mM) A 7.5 60 60 0.06 – 10 B 7.5 60 60 0.5 – 10 C 7.9 100 100 0.5 – 10 D 7.9 60 60 1.5 – 10 E 7.5 60 60 1.0 – 10 F8.5 100 100 1.0 – 100 G 8.2 500 50 – – – H 7.5 900 100 0.5 – – J 7.5 100 70 – 0.5 10 K 7.4 100 100 – 1.5 – L 9.0 100 30 1.0 – – MULTI-CORE™ Buffer (10X) = .25M Tris-Acetate (pH 7.8 at 25°C), 1M potassium acetate, 100mM magnesium acetate, 10mM DTT. Notes: 1. For each 10°C rise in temperature between 0°C and 25°C, the pH of Tris buffers decreases 0.31 pH units. 2. For each 10°C rise in temperature between 25°C and 37°C, the pH of Tris buffers decreases 0.25 pH units. ENZYME RESOURCE GUIDE Glossary Accuracy: The average number of nucleotides the polymerase incorporates before making an error. Activated calf thymus DNA: Nicked and gapped dsDNA prepared by treatment with DNase I; a substrate for many DNA polymerases. BuAdATP: 2-(p-n-butylanilino)-2′-deoxyadenosine 5′-triphosphate. Also known as butylanilino dATP. BuPdGTP: N2-(p-n-butylphenyl)-2′-deoxyguanosine 5-triphosphate. Also known as butylphenyl dGTP. dNTP: Deoxyribonucleotide 5′-triphosphate. Distributive: Enzyme dissociates from template after a single nucleotide addition. ds: Double-stranded. DTT: Dithiothreitol. Error rate: The mutation rate per base pair duplicated; accuracy is the inverse of error rate. Fidelity: A polymerase’s ability to faithfully replicate a DNA molecule. I50: Concentration of a substance that causes 50% inhibition in an enzyme’s activity. IDmin: The minimum inhibitor concentration causing inhibition of enzyme activity. ID50: See I50. kb: Kilobase or kilobase pairs. kcat : Maximum number of substrate molecules converted to products per active site per unit time. kDa: KiloDalton. Km: The Michaelis constant is the concentration of substrate at which an enzyme can convert to product at half its maximal rate. Misinsertion: A rare event in which a polymerase adds a nucleotide to the 3′-terminus of a primer that does not conform to AT and GC base pairing with the template. Misinsertion usually leads to a mutation after duplication of the cDNA. Mismatch: When bases on opposite strands of dsDNA are in the same position but do not conform to AT and GC base pairs. Mesophilic: Optimal activity at temperatures around that of a warm blooded animal (i.e., 37˚C) but includes the temperature range 15–45˚C . Nick translation: Polymerization at a DNA nick with concurrent hydrolysis of the 5′-end at the nick, so as to translate (move) the nick without a change in mass of the DNA. PCR(g): Polymerase Chain Reaction. PEG: Polyethylene glycol. PPi : Inorganic pyrophosphate. Primer: Short ssDNA that binds to a longer, complementary template strand; the 3′-terminus serves as the site of polymerization. Processive: Repetitive enzyme action without dissociation between steps; nondistributive. Promoter: DNA sequence for the initiation of RNA transcription by RNA polymerase. Read-through transcript: Describes either those RNA transcripts not properly terminated by RNA polymerase or RNA transcripts that proceed from the promoter to the end of a linear template. ss: Single-stranded. Strand displacement: Ability of a polymerase to replicate at a nick by melting the dsDNA and displacing the 5′-chain as it polymerizes a new copy of the displaced ssDNA. 5′-RACE: Rapid amplification of cDNA 5′ ends. rNTP: Ribonucleotide 5′-triphosphate. TAE: Tris Acetate EDTA . TCA: Trichloroacetic acid. Template: Complementary DNA chain providing the sequence that directs nucleotide additions to the primer strand. Terminator: DNA sequence for the termination of RNA transcription by RNA polymerase. Thermostable: The ability to withstand prolonged exposure to an elevated temperature, e.g., 95˚C. Thermophilic: Optimal activity at elevated temperatures, usually above 50–60˚C. TUNEL Assay: TdT-mediated dUTP Nick End Labeling Assay. Turnover rate (kcat): Maximum number of substrate molecules converted to product per active site per unit time. PROMEGA 34 ONE TO ORDER Phone 1-800-356-9526 Fax 1-800-356-1970 Online www.promega.com Altered Sites, fmol, LambdaGEM, pALTER, pGEM, pGEMEX, RiboClone, Riboprobe, TaqTrack, and TNT are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office. Dead End, MULTI-CORE, GeneEditor, Taq Bead, and RiboMAX are trademarks of Promega Corporation. Coomassie is a registered trademark of Imperial Chemical Industries, Ltd. DeepVent is a trademak of, and Vent is a registered trademark of New England Biolabs, Inc. GenBank is a registered trademark of U.S. Dept. of Health and Human Services. NEN is a registered trademark of duPont NEN® Research Products. Nonidet is a registered trademark of Shell International Petroleum Company, Ltd. Triton is a registered trademark of Union Carbide Chemicals and Plastics Co., Inc. Tween is a registered trademark of ICI Americas, Inc. UlTma is a registered trademark of Roche Molecular Systems, Inc. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-todate information on Promega products. Applications mentioned in Promega literature are provided for informational purposes only. Promega does not warrant that referenced applications have been tested in Promega laboratories. (a) U.S. Pat. No. 5,552,302 and other patents. Inhibitors of Angiogenin, which comprises a segment of human PRI, is the subject of U.S. Pat. No. 4,966,964 and other patents assigned to the President and Fellows of Harvard College and exclusively licensed to Promega Corporation. (b) Covered under U.S. Pat. No. 5,168,062 assigned to the University of Iowa Research Foundation. (c) U.S. Pat. No. 5,780,270. (d) Some applications in which this product may be used are covered by patents issued and applicable in certain countries. Because purchase of this product does not include a license to perform any patented application, users of this product may be required to obtain a patent license depending upon the particular application and country in which the product is used. For more specific information, please contact Promega. (e) Patent Pending. (f) U.S. Pat. No. 5,108,892. (g) The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license. (h) Licensed from Boehringer Mannheim GmbH under U.S. Pat. No. 4,804,748. (i) U.S. Pat. No. 4,766,072. (j) Usage restrictions apply to Bacterial Strains JM109(DE3) and BL21(DE3)pLysS, to the following Promega products that include these bacterial strains (pGEMEX®-1 and pGEMEX®-2 Vectors, pET-5 and pET-9 Vectors, and all pET Vector Systems) and to any derivatives thereof. Please read the statement (z) below describing these restrictions before purchasing any of these products. (k) P1290 and P1300 are covered by U.S. Pat. No. 5,256,555 and are sold under a license from Ambion, Inc. They are intended for research use only. Parties wishing to use these products for other applications should contact Ambion, Inc. (l) The method of recombinant expression of Coleoptera luciferase is covered by U.S. Pat. Nos. 5,583,024, 5,674,713 and 5,700,673. (m) U.S. Pat. No. 5,283,179 and other patents. (n) U.S. Pat. Nos. 5,492,817, 5,665,563 and other patents. (o) Manufactured for Promega Corporation by NEN® LifeScience Products under U.S. Pat. Nos. 5,047,519 and 5,151,507. (z) Usage Restrictions for the T7 Expression System The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U.S. Department of Energy and is the subject of patents and patent applications assigned to Brookhaven Science Associates, LLC (BSA). This technology, including bacteria, phage and plasmids that carry the gene for T7 RNA polymerase, is to be used for academic or nonprofit laboratory or licensed commercial research purposes only. By accepting or using the T7 expression technology you agree to be bound by the following conditions set forth by BSA. The initial purchaser may refuse to accept the conditions of this notice by returning this product and the enclosed materials to Promega unused. Academic and NonProfit Laboratories No materials that contain the cloned gene for T7 RNA polymerase may be distributed further to third parties outside of your laboratory unless the recipient receives a copy of this assurance notice and agrees to be bound by its terms. This limitation applies to Bacterial Strains JM109(DE3) and BL21(DE3)pLysS, to the following Promega products that include these bacterial strains (pGEMEX®-1 and pGEMEX®-2 Vectors, pET-5 and pET-9 Vectors, and all pET Vector Systems) and to any derivatives thereof. Commercial Laboratories A license is required for any commercial use of the T7 expression system, including use of the T7 system for research purposes or for production purposes by any commercial entity. Information about commercial licenses may be obtained from the Licensing Office, Brookhaven National Laboratory, Upton, NY 11973, Telephone: 516-344-3312, FAX: 516-344-3729. 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All Rights Reserved. Prices and specifications subject to change without prior notice. Rev.092298 Printed in USA 10/98 Part #BR075A 1998 Promega Corporation. All Rights Reserved. Prices and specifications subject to change without prior notice.