Manual - Trimgen
Contents
1.2. B 4 Transfer 6 ul of PCR products to each tube the remaining PCR products can be stored at 20 C for re test B 5 Mix the contents and spin all tubes B 6 Incubate the tubes in a thermal cycler using Program 2 Program 2 37 C for 25 min 95 C for 5 min Hold at 4 C During the clean up incubation prepare steps C1 C4 12 C STA Reaction Mutation Detection C 1 Collect one 2 ml tube and label with ST Mix the ST reagent and detection primers to make the pre mixed ST Mix The pre mixed ST Mix can be prepared using the following formula Pre mixed ST Mix KRAS ST 61 11 x 1 x1 1 of C UP samples KRAS DP 61 2 x 1 x1 1 of C UP samples One extra tube for mutant controls KRAS CTL 61 Adjustmentfor pipetting error Add reagents to the ST tube and mix gently C 2 Collect 0 2 mL strip tubes one tube for each C UP treated sample Add an extra tube for mutant controls KRAS CTL 61 and label the tubes as follows Sample 1 2 3 Extra tube for mutant controls D The KRAS CTL 61 must be run each time C 3 Transfer 13 ul of ST Mix from step C 1 into each tube C 4 Add 5yl each of C up treated controls and samples to their corresponding tube C 5 Add 2ulof KRAS CTL 61 to the CTL tube C 6 Mix the contents and spin all tubes C 7 Place the tubes into a thermal cycler and perform ST reaction using Program 3 13 D 2 D 3
3. 6 size marker The wild type peak is observed in every sample If the peak is not observed it indicates that the DNA amplification failed See troubleshooting section F 4 or the sample is 100 mutant such as mutant cell lines If sample contains mutation s the mutation s will show as an additional peak s Compare the peak size and color with the KRAS CTL 61 panel The peak size may be slightly shifted due to migration differences between capillary tubes Compare the wild type peak of the sample with the wild type peak of KRAS CTL 61 to identify the migration shift Any peak that does not match with the mutant controls will not be considered see trouble shooting F 6 Example of assay results Sample FFPE sample one section 1 x 0 5 cm 10 um DNA extraction WaxFree DNA kit 1 uL extract w as used for assay 35 37 39 4 43 45 a m o Mutation red Q61L CAA gt CTA Wild type black Mutation black Q61H CAA gt CAC Wild type black w R W D W oO N w S Mutation blue Wild type black Q61E CAA gt GAA Pa ee Pa 16 F F 2 F 3 Troubleshooting Color leak through When the sample DNA concentration is too high the ST reaction generates a strong fluorescent signal gt 5 000 rfu Fluorescence spillover will occur For example the black peak of the wild type signal may be observed in the red and or blue channels This co
4. analysis Mutation 25 40 min Time varies depending on the type of sequencer Materials Provided The Mutector KRAS Codon 61 Mutation Detection kit contains reagents enough for 32 tests 650 ul Master Mix Reagents for DNA amplification KRAS 61 PCR 50 ul PCR primer mix for amplification of KRAS Primers H gene codon 61 C UP1 20 ul Enzyme 1 for cleanup of PCR products 20 ul Enzyme 2for cleanup of PCR products C UP Buffer 430 ul Buffer for C UP reaction 430 ul Pre mixed STA reagents for detection of KRAS ST 61 H KRAS codon 61 mutations Pre mixed detection primers for KRAS KRAS DP 61 80 pl codon 61 mutations KRAS CTL 61 60 ul Mutation controls for KRAS codon 61 Loading Buffer 1000 ul Sample loading buffer with size standards 3 Light Sensitive Keep these reagents protected fromdirect light Materials required 0 2 mI PCR tubes 8 well strip tube DS 32 Matrix Standard kit Applied Biosystems Cat No 4345831 This kit is a one time calibration to setup the correct spectral channels This is required for all Mutector Il assays Equipment required Thermal Cycler Any type of thermal cycler with a 0 2 ml tube blockis acceptable for performing the assay Sequencer Applied Biosystems Genetic Analyzer Instrument Data Collection Genetic analy zer 3100 Data Collection Genetic analy zer 3700 powan Genetic analy zer 3130 3500 Data Collection Genetic analy zer 3500 Software v 1 0 Dat
5. 2014 Introduction Mutector KRAS Codon 61 Mutation Analysis Reagents are designed to detect and differentiate the following 5 mutations occurring in codon 61 of the KRAS gene Codon 61 mutations The mutation detection is performed in a single tube Each kit provides reagents enough for 32 reactions The assays products are analyzed on an Applied Biosystems Genetic Analyzer using fragmentanalysis software The kit uses Shifted Termination Assay STA technology to enrich the mutation signal and is able to accurately detect low level somatic mutations Shifted Termination Assay STA Shifted Termination Assay is a proprietary technology that uses uniquely designed primers mixtures of modified enzymes and specially synthesized nucleotides STA technology extends primers by multiple bases to increase signal strength and fragment size creating mutation peaks that are easily distinguished from wild type The enriched mutation signals are then detected by fragment analysis The STA technology can detect low level mutations often missed by sequencing Wild type j X Mutant a X STA reaction Fragment analysis Overview of Mutector Assay PCR Amplification 1 5 hours Time varies by thermal cycler used PCR Product Clean up 30 min STA reaction Mutation detection 40 min Time varies by thermal cycler used Sample Loading To Sequencer Wil ne ppe Capillary Electrophoresis Fragment
6. ECTION WITH OR ARISING F ROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT TRIMGEN IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Limited Product Warranty It is imperative that the users strictly adhere to this manual Failure to do so will void TrimGen s guarantee of this product TrimGen Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness fora particular purpose License The purchase of Mutector kit includes a limited nonexclusive license to use the kit This license does not grant rights to reproduce or modify the Mutector kit for resale or to use the Mutector kit to manufacture commercial products without written approval of TrimGen Corporation No other license expressed implied or by estoppels is granted Product Safety and Liabilities When working with the kit reagents always wear a lab coat disposable gloves and protective goggles TrimGen Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the misuse the results of use or the inability to use this product Trademarks The trademarks mentioned herein are the property of TrimGen or their respective owners Trim Gen Corporation All rights reserved Information in this document is subject to change without notice TrimGen GP06 KRAS Codon 61 Manual 10
7. Miutector a Mutation Detection Kit I KRAS Codon 61 Mutation Analysis Reagents f U f j ser Manual V1 4 j A _ Cat No GP06 32 reactions TrimGen Wivw trimgen com f d CONTENTS Introduction 4 Overview of Mutector Assay 5 Materials Provided 6 Materials Required 7 Equipment Required 7 DNA Sample Preparation 8 Sequencer Setup 8 Thermal Cycling Programs 9 Mutector Assay Protocol 10 A PCR Amplification 10 B PCR Product Clean Up 12 C STA Reaction 13 D Sample Loading 14 E Data Analysis 15 F Troubleshooting 17 Storage Upon receipt of the kit store at 20 C until use At this temperature the reagents are stable for 6 months After first use store all of reagents at 2 8 C and keep them protected from direct light At this condition the reagents are stable for 1 month Notice to Purchaser The Mutector kit is provided as research use only not for use in diagnostic procedures The purchaser must determine the suitability of the productfor their particular use TRIMGEN DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW INNO EVENT SHALL TRIMGEN BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONN
8. Program 3 1 cycle 94 C 4 min 20 cycles 94 C 20sec 60 C 30sec 70 C 20 sec Hold at 4 C During the STA reaction prepare step D1 D3 Sample Loading Add 15 ul of the Loading buffer to each well of a sequencer adapter plate Transfer 5 ul of the ST products into each well and remove any bubbles in the well Load the plate to sequencer and run the pre set Data Collection Program ref page 8 14 E 2 E 3 Data Analysis Open the analysis software GeneMapper or GeneScan Follow the instructions to add the data for analysis The instructions are provided online GeneMapper www trimgen com docs Partlll Data Analysis GeneMapper pdf GeneScan www trimgen com docs PartlV Genescan pdf Confirm results of KRAS CTL 61 mutant controls In the sample plotwindow shows graphic data find the results for the CTL K61 The CTL shows 6 peaks All peaks are located between 32 42 on the X axis zoom in on the X axis to 25 2 size marker 80 6 size marker The peak size of KRAS CTL 61 is usedas the standard for sample analysis Result for KRAS CTL 61 Peak Color Peak size interpretation Peak Color Peak Size Interpretation Red 3683 Mutation Q6iH CAA gt CAT Red 3820 mnaion Qeil CAA gt CTA Peak size may vary slightly depending on instrument polymer type and the length of capillary 15 E 4 Sample analysis Zoom in on the X axis to 25 2 size marker 80
9. a Analysis GeneMapper Softw are v4 0 or v4 1 GeneMapper Software v4 1 DNA Sample Preparation Reagents for DNA preparation are not provided with the kit Paraffin FFPE and fresh or frozen tissue samples TrimGen has developed the WaxFree DNA extraction kit especially for FFPE samples The kit uses special resins that bind and remove PCR inhibitors in the tissue extracts leaving all DNA or RNA fragments in the extract This method recovers more DNA in comparison with other extraction methods The kit has been validated in many laboratories using a variety of FFPE samples as well as fresh and frozen tissue samples WaxFree s simple procedure and high DNA yield ensures a PCR amplification success rate of gt 95 Product information WaxFree DNA for 50 samples Cat WF 50 WaxFree DNA for 100 samples Cat WF 100 DNA concentration When using a column or bead DNA extraction method adjustthe final concentration of extracted DNA to 20 80 ng ul When using TrimGen s WaxF ree DNA kit follow the user manual to perform PCR reaction Sequencer setup First time users should setup the analysis program for the ABI sequencer one time setup After setup the program can apply to all Mutector tests for data analysis GeneMapper Analysis Step I GeneMapper Setup www trimgen com docs Partl GeneMapper Setup pdf Step Il Data Collection Software Setup www trimgen com docs Partll Data Collection Setup pd
10. d label the tubes as follows Sample 1 2 3 ma LLANO OOC Neg Negative Control Pos Positive Control A 3 Transfer 19 lof PCR Reaction Mix into all of the tubes A Add 1 ulof nuclease free water to the Neg tube A 5 Add 1 pl of KRAS Codon 61 Positive Control to the Pos tube 10 A 6 Add 1 2 pl of sample DNA 20 80 ng ul to each sample tube When using TrimGen WaxFree kit for paraffin sample DNA extraction add 0 5 1 ul final extract to each sample tube Add too much sample may cause an inhibition of PCR reaction A 7 Place the PCR tubes ina thermal cycler and run Program 1 Program 1 1 cycle 94 C 5 min 35 cycles 94 C 30sec 52 C 30sec 72 C 30sec 1 cycle 72 C 5min Hold at 4 C Optional The PCR products can be verified by agarose gel electrophoresis 5 ul loading The correct band size is 120 bp The procedure can be temporarilystopped after Program 1 y The PCR products can be stored at 4 C for 2 3 days During the PCR amplification process prepare steps B1 B2 11 B PCR Products Clean Up B 1 Prepare C UP Mix C Buffer 10 uL x x1 1 of PCR tubes C UP1 0 5 uL x x1 1 of PCR tubes C UP2 0 5 uL x x1 1 of PCR tubes Mix the reagents and spin down For pipetting error B 2 Collect0 2 ml strip tubes one tube for each PCR reaction Label the tubes the same way as the PCR tubes B 3 Add 11 pl of C UP Mix to each new tube
11. e size standard is too low the software cannot detect the size standard correctly and the program will not show the graphic data Diluting the final ST product with de ionized water and reloading the sample will easilyresolve this problem The size standard may be miscalculated Check the size standard and manually correct the size standard see the 17 F 4 F 5 F 6 F 7 sequencers instruction manual Reanalyze the data after correction of the size standard No wild type peak The wild type peak is an internal control for sample DNA amplification this peak should show in allsamples If the peak is not observed it indicates that the PCR amplification failed The possible causes could be poor DNA quality low DNA concentration and or existence of PCR inhibitors in the DNA sample see page 8 for DNA sample preparation section Background noise Nomally the background of the assay is low When the peak signal is too strong over 8000 rfu and highlighted with pink color background noise may pull up as peak To resolve this issue simply dilute the final ST product with de ionized water and re load the sample A peak that does not match with any peak in Mutant Controls CTL If such peaks is detected please contact our tech supportfor further analysis In some circumstances when the sample DNA concentration is too low or the PCR did not amplify DNA properly an unusual peak will appear ina very different position m
12. f Step Ill Data Analysis Using GeneMapper www trimgen com docs Partlll Data Analysis GeneMapper pdf Important Spectral calibration is required before runnin ihe test The sequencer needs to be calibrated with the DS 32 calibration kit Applied Biosystems catNo 4345831 This is a one time calibration to set up spectral channels to collectthe test results Refer to the DS 32 Matrix standards kitto prepare the DS 32 matrix standards Runa Matrix Standard Set DS 32 5FAM JOE NED ROX to perform a spectral calibration Thermal Cycling Programs Program 1 PCR 1 cycle 94 C 5 min 35 cycles 94 C 30 sec 52 C 30 sec 72 C 30 sec 1 cycle 72 C 5 min Hold at 4 C Program 2 Clean up 37 C 25 min 95 C 5 min Hold at 4 C Program 3 EM reaction 1 cycle 94 C 4 min 20 cycles 94 C 20 sec 60 C 30 sec 70 C 20 sec Hold at 4 C Mutector Assay Protocol A PCR Amplification Thaw all reagents and keep on ice Spin down the reagents before use A negative control water is recommended to run with samples eachtime A 1 Prepare PCR Reaction Mix Master Mix 18 x 2 x1 1 of Samples KRAS 61 PCR Primers 1 x 2 x1 1 of Samples For negative and positive sample controls For pipetting error Transfer entire volume of the reagents to one tube and gently mix avoid bubble the contents This is the PCR Reaction Mix A 2 Collect0 2 mI PCR strip tubes an
13. lor spillover is caused by limitation of the instrument The leak through peak will have the exact same peak size as the original peak Because the mutation peaks have different peak size leak through will not affect data analysis The peak signal is too high The assay is set at a condition to detect mutations in a small sample such as DNA extracted from fine needle aspiration FNA sample For regular FFPE sample the assay signal may be too high to analyze peak height gt 8000 rfu cannot see the top of the peak or the peak is highlighted with pink color Diluting the final STA product with de ionized water can efficiently reduce the signal and optimize the peak height Do not dilute the assay reagents it will cause improper enzymatic reaction and generate a miss call Each laboratory has different PCR instrument s the signal intensity may vary among the laboratories first time users should define the dilution factor 1 20 times dilution Once the dilution factor is determined the assay will have consistent results Graphic data will not automaticallyshow Check the raw data If the signals from the sample and size standards are too low the capillary tube may be blocked by a bubble The sample needs to be re loaded When adding a sample to the loading plate carefully add the sample to avoid bubbles The ST products will compete with the size standard DNA to enter the capillary tube If the sample signal is too strong and th
14. ostof them are far from the wild tyoe peak Any peaks outside ofthe data interpretation zone 25 80 on x axis are not considered for analysis Mutation peak cut off For some samples a small peak maybe observed in one of the mutation positions To verify the peak you need to confirm the signal strength ofthe wild type peak If the wild type peakis too high cannotsee the top of the peak and the peak is highlighted with pink color your ST reaction is too strong and the small peak may be pull up from background noise Follow F 2 to dilute the final product of the ST reaction with de ionized water After dilution reload the sample If you can see the top of the wild type peak use the following calculation to identify the small peak Ratio Area of mutantpeak Area of wild type peak 18 F 8 If the ratio is larger than 0 06 the peakis determined to bea mutation peak the ratio does not representthe percentage of the mutation presentin the sample Otherwise the peakis a background pull up and does notindicate the presence ofa mutation in the sample Bumper peak For some samples there are peaks that show as a bumper see figure below Most of these peaks are background pull up The causes for the bumper peaks are over loading of the ST product Refer to F 2 in the Troubleshooting to dilute the final ST product Wild type The sample is over loaded Mutation Bumper peaks 19
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