Table of contents. I. Description...2. II. Principle...2. III. Kit Components...3. IV. Storage...3. V. Features...4. VI. Precautions for Operation...

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1 Table of contents I. Description...2 II. Principle...2 III. Kit Components...3 IV. Storage...3 V. Features...4 VI. Precautions for Operation...4 VII. Protocol...4 VIII.Experiment Example...6 IX. Appendix...8 X. Reference...8 XI. Related products...8

2 I. Description : CycleavePCR Core Kit is designed for exclusive use for real-time PCR * 1 with Cycling Probes for detection. Real-time PCR is a very useful technology which allows quick and quantitative reaction. Cycling Probe Technology * 2 allows highly specific detection. The combination of real-time PCR and Cycling Probe Technology achieves the accurate and quick detection of a target gene and its quantification, or detection of SNP (single nucleotide polymorphism). This kit can be used with Smart Cycler System or Smart Cycler II System * 3 (Cepheid). * 1:For real time PCR with other various probes (e.g. TaqMan, Molecular Beacon) or SYBR Green I, Premix EX Taq (Perfect Real Time) (Cat.#RR039A) or SYBR Premix EX Taq (Perfect Real Time) (Cat.#RR041A) should be used. * 2:Cycling Probe Technology and DNA-RNA-DNA chimeric nucleic acid technology are licensed by ID Biomedical Corporation. * 3:Smart Cycler System is a registered trademark of Cepheid. CycleavePCR Core Kit simultaneously carries out both PCR amplification with TaKaRa Ex Taq Hot Start Version and detection of the amplified product in real-time through Cycling Probe Technology. II. Principle : (1) PCR PCR (Polymerase Chain Reaction) is a simple and powerful method to amplify only a target gene in vitro from a tiny amount of DNA. It can amplify the target DNA fragment up to 10 6 folds in a short time by repeating the cycle composed of three incubation steps at three different temperatures; denaturation of DNA, primer annealing, synthesis of complementary chain with DNA polymerase. As this kit uses Takara's PCR enzyme efficient for Hot Start PCR, TaKaRa Ex Taq Hot Start Version, non-specific amplification deriving from mispriming or from primer-dimers before thermal cycling can be prevented and it achieves highly sensitive detection. (2) Cycling Probe Technology Cycling Probe Technology is a high-sensitive detection method utilizing a combination of chimera probe, composed of RNA and DNA, and RNase H. The specific sequence of target gene to be amplified can be detected efficiently during or after amplification by this method. As long as this probe remains intact, no strong fluorescence can be emitted because of the quenching function. When this probe forms a hybrid with the complementary sequence of amplified product, RNase H specifically cuts the RNA region of this probe, resulting in emission of strong fluorescence. By measuring the intensity of emitted fluorescence, the amount of amplified product can be monitored. Since the probe including mismatch in its probe region is not cut by RNase H, Cycling Probe Technology allows the highly specific detection which can recognize even a SNP. 2

3 Principle of Cycling Probe Technology : Quencher Q R NA Fluoresceine substance F Q F Q R Nas e H F Q F Amplified products Formation of hybrid Cut RNA part by RNaseH Increase of fluorescein intensity III. Kit Components (for 50 reactions) : x CycleavePCR Buffer 125 μl 2. dntp Mixture 2.5 mm each 150 μl 3. Mg solution 25 mm 250 μi 4. TaKaRa Ex Taq Hot Start Version 5 U/μl 12.5 μl 5. Tli RNaseH II 200 U/μl 25 μl 6. Positive Control 10 4 copies/μi 10 μl 7. Positive Control primer mix 10 μl 8. Positive Control probe (FAM) 25 x 10 μl 9. dh2o 700 μl *:The supplied Positive Control is a plasmid. Its copy number is calculated based on the OD260 value and it does not necessarily reflect the real molecule number. Reagents and Instruments Required but Not Supplied in the Kit 1. Thermal Cycler for real time PCR Smart Cycler System (Cepheid) Smart Cycler II System (Cepheid) 2. Smart Cycler reaction tube (Cepheid) 3. PCR primer * 1 4. Cycling probe * 1 5. Micropippets and Micropippet tips (autoclaved prior to use) * 1:Please refer to IX. Guideline for designing primer for the method to design PCR primers. Takara can design and synthesis of PCR primer or Cycling Probe of a customer interest as custom service. Takara also offer the service to optimize the reaction condition of real time PCR with Cycling Probe and Smart Cycler System. Please contact TAKARA BIO INC. IV. Storage : - 20 (for shipping and storage) 3

4 V. Features : (1) The combination of real time PCR and cycling probe technology allows quick and accurate result of PCR reaction. This combination can be utilized to establish a detection system for SNP analysis which discriminates the difference in single base. (2) This kit uses Takara's PCR enzyme efficient for Hot Start PCR, TaKaRa Ex Taq Hot Start Version. As this enzyme is optimized for use in real time PCR, this product offers high amplification efficiency and highly sensitive detection. VI.Precautions for Operation: Please ensure to read through the following precautions prior to starting the protocol. (1) It is recommended to prepare Master Mix (the mixture of dh2o, buffer, enzymes, etc.) in the amount for several reactions. The preparation of Master Mix minimizes the loss by pipetting, dispensing the reagents, and mixing frequency so that it can allow accurate dispensing or reagent per reaction. Accordingly the variance in experiment can be minimized. (2) The enzymes such as TaKaRa Ex Taq Hot Start Version, Tli RNase H II should be mixed genltly, without gently, without generating bubbles. Prior to pipetting, please centrifuge them gently to drop the reagents on the wall to a tube bottom. As the enzymes are highly viscous containing 50 % glycerol, pipetting should be carried out carefully. (3) The enzymes should be stored at - 20 before use, and should be returned to - 20 freezer immediately after used. (4) For dispensing the reagents, a new disposable tips should be used to minimize contamination among samples. VII. Protocol : 1. Prepare the PCR reaction mixture on ice. Prepare the following reaction mixture in the volume of required reaction number and additional few. Then dispense them into Smart Cycler tube in 24 μl each. < One reaction > Reagent 10 x CycleavePCR Buffer 2.5 μl 1X dntp Mixture (2.5 mm each) 3 μl 0.3 mm Mg solution (25 mm) 5 μl 5 mm PCR Forward Primer (20 μm) 0.25 μl 5 pmol PCR Reverse Primer (20 μm) 0.25 μl 5 pmol Cycling Probe (5 μm) * 1 μl Tli RNase H II (200 U/μl) 0.5 μl 100 U TaKaRa Ex Taq Hot Start Version (5 U/μl) 0.25 μl 1.25 U dh2o μl Total 24 μl *:Cycling Probe is generally used in 5 pmol per reaction, but the amount should be adjusted depending on the signal intensity. When used with Smart Cycler, it should be adjusted to have the fluorescence intensity of

5 2. Add 1 μl of template into the Smart Cycler tubes prepared at 1. The template can be added in more volume than 1 μl. In this case, the volume of dh2o should be adjusted depending on the template volume. 3. Start the reaction. Gently centrifuge the tubes in a centrifuge for Smart Cycler. Load the tubes on a Smart Cycler and start the reaction. Refer to the following table for setting the reaction parameter. Step Temp. Time Detection Remark Initial denaturation ~ 30 sec. OFF When the template is genome DNA, heat denaturation for 30 sec., or possibly 1 min depending on a case, is necessary. (The reaction may not be stable under the denaturation over 1 min.) The template of less than 500 bp might not require the initial denaturation. Denaturation 95 5 sec. OFF As the amplified size of the target in real time PCR is generally less than 500 bp, denaturation of 3-10 sec. is sufficient. Annealing ~ 20 sec. OFF Extension ~ 15 sec. ON Cycle number 30 ~ 50 cycles When non-specific product is generated or amplification efficiency is low, optimization of annealing temperature can work to improve. Longer annealing time can sometimes improve the amplification efficiency. When the amplified size is around 100 bp, sec. is adequate. Then the size Is longer than 100 bp, the time can be extended by 5 sec. per 100 bp. Smart Cycler terminates the reaction at the point when the amplified product is detected. This function can be utilized for more quick analysis. * *:This function is available in Smart Cycler Software Version 2.0. Although it is not originally equipped in Smart Cycler Software Version 1.2, it can be available by upgrading with Smart Cycler Software Version 2.0 Upgrade Kit. This operation does not require the upgrade of the instrument itself. 4. After the reaction process terminates, perform analysis. After the reaction process terminates, confirm the amplification curve and perform analysis. Please refer the instruction manual of Smart Cycler system and VIII. Experiment Example for the analysis method in Smart Cycler. 5

6 VIII. Experiment Example : Confirmation of CycleavePCR reaction using Positive Control (Smart Cycler II) The supplied Positive Control allows to verify if the operation is followed correctly. 1. PCR reaction : Prepare the reaction mixture including Positive Control Primer mix and Positive Control probe in each 1 μl, by following the protocol described in VII. Protocol. The composition of reaction mixture is as below. 10 X CycleavePCR Buffer 2.5 μl dntp Mixture (2.5 mm each) 3 μl Mg solution (25 mm) 5 μl Positive Control primer mix 1 μl Positive Control probe 1 μl Tli RNase H II (200 U/μl) 0.5 μl TaKaRa Ex Taq Hot Start Version (5 U/μl) 0.25 μl Positive Control 1 μl dh2o μl Total 25 μl 2. Set the reaction parameter as shown below. 6

7 3. Example of Positive Control The following result is expected in case of a reaction with Positive Control. Amplification curve 2nd derivative 7

8 IX. Appendix : Guideline for Designing Primer Designing primers specific to a reaction is essential to achieve highly sensitive detection via real-time PCR. Please refer to the following guideline for primer desiging. Amplified Product Amplified size Tm < bp is the most recommended Fragments of up to 300 bp can be amplified efficiently. Higher temperature may lower the reaction efficiency. Primer Lengt 17 ~ 25 mer GC content 40 ~ 60 % Tm 55 ~ 65 Sequence Complimentarity Specificity Tm values of Forward primer and Reverse primer must not differ largely. 3' termini should not have the sequence having three G or C in row. 3' termini should not be T The inside sequence of the primer should not be the same in more than 2 bases as that of 3'-termini. Perform BLAST search to confirm the primer specificity. * It can lead to low specificity in annealing, thus non-specific product would be formed, e.g. primer dimer. It can lead to mismatch annealing. The specificity would be low. It can form primer dimer. BLAST/ Takara can design and synthesis of PCR primer or Cycling Probe of a customer' interest as custom service. Takara also offer the service to optimize the reaction condition of real time PCR with Cycling Probe and Smart Cycler System, such as SNP detection system. Please contact TAKARA BIO INC. X. Reference : F. Bekkaoui et al. (1996) BioTechniques 20, XI. Related products : Smart Cycler System (Cepheid) Smart Cycler II System (Cepheid) 8

9 NOTE : This product is intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc. Takara products may not be resold or transfered, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please call at or contact from our website at NOTICE TO PURCHASER: LIMITED LICENSE [P1] PCR Notice Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser's own internal research. No right under any other patent claim (such as the patented 5' Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. [L4] Quencher This product is for measurement of amplification detection for research use only in life sciences research, industrial and environmental testing (including food industry, but excluding bio-terrorism and bio-warfare), non-human animal diagnostic testing, forensic testing and providing services to third parties who do not use the services for the purpose of (a) providing patient management or care, and in which the results of such services are not included in patient records and (b) providing bio-terrorism or bio-warfare testing; and specifically excluding, without limitation: (I) any human clinical, therapeutic or diagnostic uses and animal clinical and therapeutic uses (including any use of the results of any testing performed with any product for patient management or care, or the use of the results of the services for patient management or care) and (II) any research or services where the results of any test or assay are used for patient management, care or otherwise in making therapeutic or treatment decisions for a patient. A portion of this product is subject to proprietary rights of Epoch Biosciences, Inc. and are made and sold under license from Epoch Biosciences, Inc. under the patents and patent applications (US , WO , WO , US10/113,445, US09/876,830 and corresponding patents issued in other countries). There is no implied license for commercial use with respect to this product. A license must be obtained directly from Epoch Biosciences, Inc. with respect to any proposed commercial use of this product, and "commercial use" includes but is not limited to (A) the sale, lease, license or other transfer of this product or any material derived or produced from it, (B) the sale, lease, license or other grant of rights to use this product or any material derived or produced from it, and (C) the sale, lease, license or other transfer of kits which include this product. [L8] Cycling Probe Technology Sold under license from ID Biomedical Corp. [L15] Hot Start PCR Licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries. [M40] Thermostable RNase H This product is the subject of the pending U.S. patent application and its foreign counterparts. [M45] RNase-resistant Cycleave probe This product is the subject of the pending Japanese patent application. [M46] ICAN -Cycleave and PCR-Cycleave This product is the subject of the pending Japanese patent application. [M57] LA Technology This product is covered by the claims 6-16 of U.S. Patent No. 5,436,149 and its foreign counterpart patent claims. * U.S. Patent 5,436,149 for LA Technology owned by TAKARA BIO INC. Licensed under U.S. Patent 5,338,671 and 5,587,287 and corresponding patents in other countries. Phone: Fax: