Cat. # R100A. For Research Use. EpiScope MSP Kit. Product Manual. v1103da

Transcription

1 Cat. # R100A For Research Use EpiScope MSP Kit Product Manual

2 Table of Contents I. Description... 3 II. Principle of MSP... 3 III. Kit Components... 4 IV. Materials Required but not Provided... 4 V. Storage... 4 VI. Precautions... 4 VII. Protocol... 5 VIII. Troubleshooting... 7 IX. Experimental Example... 7 X. Quality Test... 8 XI. Related Products URL:

3 I. Description EpiScope MSP Kit is a PCR reagent designed exclusively for methylation-specific PCR (MSP). A specific enzyme combined with an optimized buffer allows MSP analyses using a bisulfitetreated DNA template containing uracil. This kit provides a higher amplification efficiency and greatly improved ability to differentiate methylated/unmethylated compared with traditional PCR reagents. The reaction system has been optimized for real-time monitoring using SYBR Green I *1, and makes it possible to perform both real-time PCR and endpoint detection under the same reaction conditions. *1: TAKARA BIO is under a license agreement with Molecular Probes Inc. for the use of SYBR Green I as a reagent for research purposes. SYBR is a registered trademark of Molecular Probes Inc. II. Principle of MSP The first step is to identify a nucleotide region whose sequence is subject to change by bisulfite treatment depending on the methylation status of the CpG sequence. Next, design two primers for this region, one for methylated CpG DNA (M primer) and the other for unmethylated DNA (UM primer). The last step is PCR amplification. Figure 1. Principle of MSP URL: 3

4 III. Kit Components [for 200 x 50 µl reactions] (1) 2X MSP Buffer (Mg 2+ plus, dntp plus) *1 1 ml x 5 (2) MSP Enzyme 240 µl (3) 100X SYBR Green I 100 µl (4) ROX Reference Dye (50X conc.) *2 200 µl (5) ROX Reference Dye II (50X conc.) *2 200 µl *1: The Mg 2+ concentration (2X) is 4 mm, and the dntp concentration (2X) is 400 µm). *2: This component is to be used for analyses using a device that corrects fluorescent signals between wells such as the real-time PCR device by Life Technologies. Please use ROX Reference Dye for Applied Biosystems StepOnePlus Real-Time PCR System or ROX Reference Dye II for 7500 and 7500 Fast Real-Time PCR System. This component is not required when using a real-time PCR device such as Thermal Cycler Dice Real Time System II (Cat. #TP900) or an ordinary PCR device for electrophoretic analysis. IV. Materials Required but not Provided 1. Reagents - Primers for PCR - Sterile distilled water 2. Materials - Special reaction tubes or plates - Micropipettes and tips (autoclave treated) - Gene amplification system for real time PCR (authorized instruments) V. Storage -20 Note: It is important to protect 100X SYBR Green I from light. VI. Precautions (1) Place all reagents, except 100X SYBR Green I, on ice when preparing the reaction mixture. 100X SYBR Green I will freeze on ice. Keep it at room temperature protected from light. (2) Use fresh disposable tips to avoid any potential contamination between samples when preparing or dispensing reaction mixtures. 4 URL:

5 VII. Protocol 1. Primer design We recommend using a primer design tool that is specific for bisulfite-treated sequences. The following design tools are available online, free of charge. (For specific operating instructions, please refer to the help section of each tool.) The optimum size of amplification products is between 80 and 150 bp (amplifications of up to 200 bp are possible). MethPrimer BiSearch URL: 5

6 2. PCR reaction composition (50-µl reaction) The reaction composition is the same for real-time PCR (quantitative analyses) and endpoint detection (to confirm the presence or absence of amplification by electrophoresis). Note: Even for endpoint detections, be sure to add 100X SYBR Green I. Amount Final conc. 2X MSP Buffer 25 µl 1X PCR forward primer 15 pmol 0.3 µm PCR reverse primer 15 pmol 0.3 µm 100X SYBR Green I 0.5 µl 1X MSP Enzyme 1.2 µl ( ROX Reference Dye(50X)or 1 µl Dye II(50X) 1X) * 1 Template <5 µl dh2o (sterile distilled water) up to 50 µl * 2 *1: This component is to be used for analyses using a device that corrects fluorescent signals between wells such as the real-time PCR device by Life Technologies. Please use ROX Reference Dye for StepOnePlus or ROX Reference Dye II for 7500 and 7500 Fast Real-Time PCR System. This component is not required with Thermal Cycler Dice Real Time System II; nor is it required for endpoint detection by electrophoresis. *2: Please change the reaction volume appropriately in accordance with the recommended volume for each PCR device. 3. PCR condition. The PCR condition is the same for real-time PCR and endpoint detection sec sec sec cycles 72 1 min.(up to 200 bp) Melting-curve analysis (for real-time PCR) Note: The MSP enzyme supplied in this kit is a hot start PCR enzyme that uses an anti-taq antibody that inhibits polymerase activity. Please do not perform the 5- to 15-minute activation at 95 before PCR reaction that is required with other companies chemically modified hot start PCR enzymes. Unnecessary heat treatment tends to reduce enzyme activity and affect amplification efficiency. Even for the initial denaturation of template, 95 for 30 sec is generally sufficient. After reaction completion, perform the required analysis for real-time PCR or an agarose electrophoresis for endpoint detection. After electrophoresis, stain the gel in the usual manner with a stain such as ethidium bromide. 6 URL:

7 VIII. Troubleshooting This product's reaction system is designed to provide a high reactivity and to differentiate methylated/unmethylated DNA. If the result is less than desirable, review the primer concentration or the PCR condition in accordance with the following procedure. <No amplification, poor reactivity> Raise the primer concentration Lower the annealing temperature Increase the number of cycles up to 45 <Poor methylated/unmethylated differentiation, significant non-specific amplification> Lower the primer concentration Raise the annealing temperature Reduce the number of cycles IX. Experimental Example 1. MSP for the promoter region of each of the CDH1, CDKN2A and MLH1 genes. Method: With bisulfite-treated methylated HeLa genome and native HeLa genome as the template (30 ng/25 µl reaction system for each), MSP was performed for the promoter region of each of the CDH1, CDKN2A and MLH1 genes. Result: Comparable results were obtained with real-time PCR and endpoint detection. With the native HeLa genome, the CpG regions of CDH1are methylated, but the CpG regions of CDKN2A and MLH1 are unmethylated. EpiScope Methylated HeLa gdna Native HeLa gdna CDH1 CDKN2A MLH1 : M primer (primer for detecting methylated DNA) : UM primer (primer for detecting unmethylated DNA) Figure 2. Detection with Thermal Cycler Dice Real Time System II, a real-time PCR device URL: 7

8 EpiScope Methylated HeLa gdna 1 M U 2 M U 3 M U Native HeLa gdna 1 M U 2 M U 3 M U (primer) 1: CDH1 2: CDKN2A 3: MLH1 Apply 5 µl Use 3% gel Figure 3. Endpoint detection. X. Quality test Excellent amplification and a high level of methylated/unmethylated differentiation (ΔCq 10) were confirmed in real-time PCR reactions (amplification products: 104 bp and 108 bp) by Thermal Cycler Dice Real Time System II, with CDH1 region-containing methylated or unmethylated model plasmid as the template. XI. Related products MethylEasy DNA Bisulphite Modification Kit (Cat. #ME001) MethylEasy High Throughput Centrifuge Method (Cat. #MEHT002) MethylEasy High Throughput Vacuum Method (Cat. #MEHT003) MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (Cat. #ME002) EpiScope Methylated HeLa gdna (Cat. #3520) Thermal Cycler Dice Real Time System II (Cat. #TP900)* * Not available in the United States or Europe. 8 URL:

9 NOTICE TO PURCHASER: LIMITED LICENSE [P5] 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, 5,677,152, 5,773,258, 5,407,800, 5,322,770, 5,310,652, 5,994,056, 6,171,785, 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 apparatus or system claims in US Patent No. 6,814,934) 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 [L11] SYBR Green I This product is covered by the claims of U.S. Patent No. 5,436,134 and 5,658,751 and their foreign counterpart patent claims. Takara PCR products containing SYBR Green I are sold under license from Molecular Probes Inc. only for the usage in real-time PCR for internal research purpose. These products are not to be used for the purpose such as; providing medical, diagnostic, or any other testing, analysis or screening services or providing clinical information or clinical analysis in return for compensations. [L15] Hot Start PCR Licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries. [L46] SYBR/Melting Curve Analysis The purchase of this product includes a limited, non-transferable license for all fields other than human or veterinary in vitro diagnostics under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein. URL: 9

10 [L51] MSP A license under U.S. Patents 5,786,146, 6,017,704, 6,265,171 and 6,200,756 or their foreign counterparts, owned by The Johns Hopkins University, is required to practice methylation-specific polymerase chain reaction and related processes ("MSP") described in said patents. The purchase price of this product includes limited, nontransferable rights to use only this amount of the product to practice MSP and related processes described in said patents solely for the internal research activities of the purchaser. No right to any other application, including any in vitro diagnostic application for commercial purposes or clinical trial testing application, or to perform or offer commercial services of any kind using MSP, including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration, is hereby conveyed or granted by implication or estoppel. Further information on obtaining licenses to practice MSP may be directed to the Licensing Department, MDxHealth S.A., Tour 5 GIGA niveau +3, Av. de l Hopital 11, 4000 Liege, Belgium. [L52] Rox Reference Dye (Research Field) Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser s own internal research. No other patent rights are conveyed expressly, by implication, or by estoppel. 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. 10 URL:

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12 NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc. Takara products may not be resold or transferred, 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 contact us by phone at or from our website at 12 URL: