Table of content. 1. Description Component Specification Optimization of parameters Extension time...
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1 Table of content 1. Description Component Specification Optimization of parameters Extension time Enzyme concentration Template DNA Primer dntp and Mg Condition of thermal cycling General reaction mxture for PCR Preparation of reaction mixture High fidelity Trouble shooting Note
2 1. Description Pyrobest TM is an α-type thermostable DNA polymerase derived from Pyrococcus sp., which possesses an associated 3' 5 exonuclease (proofreading) activity. Pyrobest TM is a most suitable enzyme for high-fidelity amplification thanks to the high proofreading activity. Generally it is considerd that α-type DNA polymerase is more difficult to use for PCR reaction than Pol I-type DNA polymerase (Taq polymerase etc.), because the optimum reaction condition of α-type DNA polymerase is relatively limited. But Pyrobest TM is able to offer productivity as well as Taq polymerase, as a result of the buffer optimization. At least 6kbp or 12kbp can be amplified from human genomic DNA template or λ-dna template using Pyrobest TM. Two advantages for PCR are supplied with Pyrobest TM ; high fidelity and good productivity. 2. Component Pyrobest TM DNA Polymerase (5 units/µl ) 25 µl 10 x Pyrobest TM Buffer II ( Mg 2+ plus, 10 mm ) 500 µl dntp Mixture ( 2.5 mm each ) 400 µl 3. Specification Thermostability ; Residual activity after incubation for 1hour at 95 C is over 90%. Amplification length achieved; Human genomic DNA 6 kbp and longer λdna 12 kbp and longer Reverse transcriptase activity; not detected Associated exonuclease activity ; 3' 5' + 5' 3' not detected Terminal of amplified fragment ; blunt-ended The thermostable DNA polymerases have generally TdT activity and add an extra nucleotide ( in particular, A) at the 3'-end of PCR puroduct. But the TdT activity is very weak in Pyrobest TM. As most of PCR products amplified with Pyrobest TM are blunt-ended, they can be applied to cloning into blunt-ended vectors. (If necessary, phosphorylate before cloning.) Optimum reaction temperature ; around 75 C Fidelity ; 10-times accurate than Taq polymerase The optimum reaction temperature of Pyrobest is 75 C, higher than that of Taq polymerase, and the activity at 40 ~ 50 C is only 2 ~ 9 % at 75 C while the activity at 40 ~ 50 C of Taq DNA ploymerase is 15 ~ 30 % 2
3 Fig.1 Relative activity (%) TaKaRa Taq Pyrobest DNA Polymerase Temperature ( C) 4. Optimization of PCR parameters It is important to optimize each PCR parameter to bring out all the talents in Pyrobest TM and lead success of PCR. 4-1.Extention time 1 ~ 2 min/ kbp (at 68 ~ 72 C) Note: 1) It is necessary to consider the proceeding speed (Pyrobest TM ; about 25 bases/sec) and the degradation of DNA by 3' 5' exonuclease activity. 2) Longer extension time may cause diffusely smeared electrophoresis bands both in shuttle PCR and in three step PCR. 4-2.Enzyme concentration Optimum concentration of Pyrobest TM 1.25 U/50 µl PCR Optimum concentration may change according to purity and amount of template DNA and amplification size Template DNA Optimum amount of template DNA are shown as below. Human genomic DNA λdna Plasmid DNA 50 ng- 500 ng /50 µl PCR 100 pg - 10 ng /50 µl PCR 10 pg pg /50 µl PCR 3
4 For more accurate amplification, it is recommended important to decrease the number of PCR Cycles and to use an sufficient amount of template DNA. Optimum amount of template DNA may change according to purity of templates Primer Optimum concentration 0.2 ~ 2 µm Primer size 25 mer ~ 30 mer 4-5. dntp and Mg 2+ Note: 1) In case of Pyrobest TM, primers may be degradated by the 3' 5' exonuclease activity. Therefore higher concentration and longer length of primers may bring on better results. 2) S-Oligo primers (3'-S primer), difficult to degradate by 3' 5' exonuclease, may raise the reactivity. 3) Degenerated primers are available for amplificationwith Pyrobest TM, but primers containing inosine are not suitable. As dntp chelates Mg 2+, high concentration of dntp lowers the effective concentration of Mg 2+. Mg 2+ 1 mm ( final conc. ) dntps 200 µm each( final conc. ) Note: 1) Excess Mg 2+ leads non-specific reaciton, and lack of Mg 2+ leads low reactivity. The presence of chelating reagent ( ex. EDTA ) decreases effectiveconcentration of Mg 2+. Concentration of Mg 2+ should be higher than total concentration of dntps. 2) The concentration of each dntp should be set at the same level. If not, misincorporation error will occur. 3) In case of Pyrobest TM, using of dutp instead of dttp lowers the reactivity Conditions of thermal cycling Initial denaturation Generally, even when genome DNA is used as template, the initial denaturation for 1 min at 94 C is sufficient 2 step or 3 step It is recommended to use 2 step PCR(Shuttle PCR). In case that Tm value of primer is low and 2 step PCR does not work properly, try with 3 step PCR. 4
5 <Shuttle PCR> 98 C 1 ~ 10 sec. 68 C 1 min/kbp 25 ~ 30 cycles <Three Step PCR> 98 C 1 ~ 10 sec 55 C 30 sec. ~ 1 min. 25 ~ 30 cycles 72 C 1 min/kbp Note: 1) For high-fidelity amplification, it is important to decrease the reaction cycles and to use an appropriate amount of template DNA. 2) To minimize the enzyme inactivation and the damage of template DNA, shorter denaturation time is better. Denature contition varies depending on a kind of thermal cycler and tube to be used. It is recommended for 10 ~ 30 sec. at 94 C or 1 ~ 10 sec. at 98 C. 3) It is recommended that the annealing temperature is setted near the melting temperature (Tm) of primers, for example, at 5 C below the Tm. 4) Touch down PCR is also effective General reaction mixture for PCR (total 50 µl) Template DNA < 500 ng Pyrobest DNA polymerase (5 units/µl) 0.25 µl 10X pyrobest Buffer II 5 µl dntp Mixture (2.5 mm each) 4 µl Primer µm ~ 2 µm (final conc.) Primer µm ~ 2 µm (final conc.) Sterilized distiled water up to 50 µl 5. Preparation of reaction mixture All reagents should be placed on ice after thawed. Prepare the reaction mixture on ice to minimize non-specific amplification which is caused by misannealing of primers. Add the reagents into a reaction tube in the order listed as below *. After adding the all reagents, mix gently by pipetting. Preparation order 1. Sterilized distilled water 2. 10X Pyrobest TM Buffer II 3. dntp Mixture 4. Template DNA 5. Pyrobest TM 6. Primer 1 7. Primer 2 Start thermal cycling as soon as the reaction mixture is prepared. 5
6 *Note : This preparation order should be strictly followed. Pyrobest TM must be added after adding dntp Mixture into a tube. In case there is no dntp Mixture in a tube, primers may be digested by the 3' 5' exonuclease activity of Pyrobest TM. Annealing/Extention reaction(or response)(2 step), Extention reaction(or response)(3 step). Considering the reaction speed of Pyrobest(about 25 base/sec) and the degradation of PCR product by its 3' 5' exonuclease activity, the extension time is generally set at 1 ~ 2 min/1kbp at 68 ~ 72 C. In most cases, the extension time of 1 min/ 1kbp is sufficient. Avoid excess longer extension time. Touch down PCR is also effective. This is a method to lower the annealing temperature by each cycle. In case of high annealing temperature, specific amplification is achieved, though the amplification efficiency is low. Then the amplified products are efficiently amplified at lower annealing temperature in next amplification step. Using this method, it is possible perform specific amplification by avoiding the occurrence non-specific amplification Extension after thermal cycling There is no need for the final extension step(eg. 10 min at 72 C) after PCR cycle which is often applied the reaction with Taq-type enzyme. It could rather cause smeared electrophoresis bands in electrophoresis. 6. High fidelity The amplification fidelities of TaKaRa Taq TM, Pyrobest TM and Pfu DNA polymerase were compared by Cline s method and Kunkel s method. Each relative fidelity against TaKaRa Taq TM is shown in Figure 2. The fidelity of Pyrobest TM was approximately 10-fold higher than that of TaKaRa Taq TM. Thermostable DNA polymerases generally have TdT activity and add an extra nucleotide overhang(especially, A) at the 3'-end of PCR product. But the TdT activity is very weak in Pyrobest, so most PCR products obtained with Pyrobest are blunt-ended. They can be applied to cloning into blunt-ended vectors.(phosphorylation before cloning is recommended depending on an used vector.) Fig.2 Taq Pyrobest TM Pfu Fidelity (relative value )
7 7. Trouble shooting No amplification or Low productivity (1) Extension time may be short. Set at 2 min. / kbp at 68 ~ 72 C. (2) Annealing temperature may be high. Decrease the temperature at 2 C intervals, or perform touch down PCR procedure. (3) Annealing time may be short. Set at 1 ~ 2 min. (4) GC contents of template may be high, or a template may have highly complex secondary structure. Raise denaturing temprature or extend time. 98 C, 1 ~ 10 sec. or 94 C, 10 ~ 30 sec. (5) Primer is not suitable. Increase the purity of primer. Adjust GC content of primer around 50 %. Prepare a longer primer. ( 25 ~ 30 mer ) Design primers not to be complementary each other at 3'-end. Use a 3' -terminal S- Oligo primer (6) Primer concentration is low. Prepare the final concentration at µm. (7) Denaturing condition may be inappropriate. 98 C 1 sec. ~ 10 sec. or 94 C 10 sec. ~ 30 sec. (8) Insufficient concentration of Mg. Determine the optiomum Mg concentration in the range up to 2mM. In this case it is necessary to take into account the concentration of dntp and Primer, and the addition of EDTA (9) Purity of template may be low. Purify it again. 7
8 (10) Amount of template may be insufficient. Apply an appropriate amount of template. Human genomic DNA ~ 500 ng / 50 µl PCR λdna ~ 10 ng / 50 µl PCR Plasmid DNA ~ 100 pg / 50 µl PCR (11) Number of PCR cycles may be insufficient. Perform PCR at ~ 40 cycles. (12) Preparation order is not corrrect. Add Pyrobest TM into a reaction tube after addition of dntp mixture. (13) Amount of enzyme may be insufficient. Use enzyme at ~ 1.25 U/50 µ l PCR. Extrabands appear (1) Annealing temperature may be low. Raise it at 2 C intervals. Perform touch down PCR. (2) Amount of template DNA may be excess. Apply an appropriate amount of template. Human genomic DNA ~ 500 ng / 50 µl PCR λdna ~ 10 ng / 50 µl PCR Plasmid DNA ~ 100 pg / 50 µl PCR (3) Number of PCR cycles may be excess. Perform PCR at cycles. (4) Amount of primer may be excess. Use primer at 0.2 ~ 2 µm ( final conc. ). (5) Primer may be long. Design primers of less than 30 mer. 8
9 Smear occurs (1) Extension time may be long. Set at 1 min. / kbp at 68 C ~ 72 C. (2) Number of PCR cycles may be excess. Perform PCR at 25 ~ 30 cycles. (3) Amount of template DNA may be excess. Apply an appropriate amount of template. Human genomic DNA ~ 500 ng / 50 µl PCR λdna ~ 10 ng / 50 µl PCR Plasmid DNA ~ 100 pg / 50 µl PCR (4) Amount of enzyme may be excess. Use enzyme at ~ 1.25 U / 50 µl PCR. Sometimes it's better to change the enzyme amount depending on amplification size, the purity, and amount of the template. 8. Note For research use only. Not for use in diagnostic or therapeutic procedures. 9
10 NOTICE TO PURCHASER: LIMITED LICENSE A license under U.S. Patents 4,683,202, 4,683,195 and 4,965,188 or their foreign counterparts, owned by Roche Molecular Systems, Inc. and F.Hoffmann-La Roche Ltd. ( Roche ), has an up-front fee component and a running-royalty component. The purchase price of this product includes limited, nontransferable rights under the running-royalty component to use only this amount of the product to practice the Polymerase Chain Reaction ( PCR ) and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the upfront fee component. Rights to the up-front fee component must be obtained by the end user in order to have a complete license. These rights under the up-front fee component may be purchased from Applied Biosystems or obtained by purchasing an Authorized Thermal Cycler. No right to perform or offer commercial services of any kind using PCR, including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration, is hereby granted by implication or estoppel. Further information on purchasing lisenses to practice the PCR Process may be obtained by contacting the Director of Licensing at Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California Phone: Fax:
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