User Manual V1.1
Contents
1. Introduction Mutector NRAS kit is designed to detect 12 mutations occurring in codons 12 and 13 of NRAS gene Codon 12 Codon 13 G12S GGT gt AGT G13S GGT gt AGT G12R GGT gt CGT G13R GGT gt CGT G12C GGT gt TGT G13C GGT gt TGT G12D GGT gt GAT G13D GGT gt GAT G12A GGT gt GCT G13A GGT gt GCT G12V GGT gt GTT G13V GGT gt GTT 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 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 i Codon 12 Codon 13 Time varies by thermal cycler used Sample Loading To Sequencer Wild type Wild type yP a aa taa Capillary Electrophoresis mutation Fragment analysis Muta2. axis are not considered for analysis Mutation peak cut off For some samples a small peak may be observed in one of the mutation positions To verify the peak you need to confirm the signal strength of the wild type peak If the wild type peak is too high cannot see 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 mutant peak Area of wild type peak If the ratio is larger than 0 06 the peak is determined to be a mutation peak the ratio does not represent the percentage of the mutation present in the sample Otherwise the peak is a background pull up and does not indicate the presence of a 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 20
3. of PCR tubes C UP2 1 0 uL x x1 1 of PCR tubes Mix the reagents and spin down For pipetting error B 2 Collect 0 2 ml strip tubes one tube for each PCR reaction Label the tubes the same way as the PCR tubes B 3 Add 12 ul of C UP Mix to each new tube 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 two 2 ml tubes and label one tube with ST12 and the other tube with ST13 Prepare ST mixes as following Tube ST12 ST12 mix for codon 12 NRAS ST 12 11 x 1 x1 1 of Samples NRAS DP 12 2 x 1 x 1 1 of Samples For CTL 12 Adjustment for pipetting error Add the reagents to the tube and mix gently Tube ST13 ST13 mix for codon 13 NRAS ST 13 11 x 1 x 1 1 of Samples NRAS DP 13 2x 1 x 1 1 of Samples For CTL 13 Adjustment for pipetting error Add the reagents to the tube and mix gently C 2 Collect 0 2 mL strip tubes one tube for each C UP treated sample One set for codon 12 ST12 set and another set for codon 13 ST13 set Add_an extra tube for each set ST12 for C12 and ST13 for 13
4. AS PCR Primers 1x 2 x 1 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 Collect 0 2 ml PCR strip tubes and label the tubes as follows Sample 1 2 3 Neg Negative Control Pos Positive Control A 3 Transfer 19 ul of PCR Reaction Mix into all of the tubes A 4 Add 1 ul of nuclease free water to the Neg tube A 5 Add 1 pl of NRAS 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 pl final extract to each sample tube Add too much sample may cause an inhibition of PCR reaction A 7 Place the PCR tubes in a thermal cycler and run Program 1 Program 1 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 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 temporarily stopped 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 x 1 1 of PCR tubes C UP1 1 0 uL x x1 1
5. CONNECTION WITH OR ARISING FROM 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 for a 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 TrimGen Corporation All rights reserved Information in this document is subject to change without notice TrimGen KRAS GP18 manual 04 2015
6. Miutector a Mutation Detection I NRAS Mutation Analysis Reagents Codons 12 and 13 f User Manual V1 1 f ri Cat No GP18 32 reactions TrimGen P t www trimgen com f pri 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 18 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 product for 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 IN NO 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
7. and label the tubes as follows C up treated controls and samples mzs ARANO ODA ae GOCOOOOO Extra tube for mutant controls al The NRAS CTL 12 and CTL 13 must be run each time 13 C 3 C 4 C 5 C 6 C 7 C 8 C 9 Transfer 13 ul of ST12 mix from step C 1 into all tubes in ST12 set Transfer 13 ul of ST13 mix from step C 1 into all tubes in ST13 set Add 5uyl each of C up treated controls and samples to their corresponding tube in both ST12 and ST13 sets Add 5yl of CTL 12 to the C12 tube Add 5yl of CTL 13 to the C13 tube Mix the contents and spin all tubes Place the tubes into a thermal cycler and perform ST reaction using Program 3 Program 3 1 cycle 94 C 4 min 20 cycles 94 C 45 sec 60 C 20 sec 70 C 20 sec Hold at 4 C During the STA reaction prepare step D1 D3 D 2 D 3 Sample Loading Add 15 pl of the Loading buffer to each well of a sequencer adapter plate Transfer 5 pl 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 Data Analysis The NRAS Controls Codon 12 and Codon 13 represent the mutation patterns color and size Use these controls as standards to identify the peaks present in the test samples Results for NRAS Controls Codon 12 8 peaks will be presented as following Peak Color Mutation Nucleoti
8. d as mutations The peak size between the control and the sample panel may slightly shifted due to migration differences between individual capillary tubes Example results of clinical FFPE samples for codon 12 analysis 32 34 36 38 40 a 44 4B 43 50 52 54 f 6000 soot 6125 Detected 4000 f 3000 2000 f 1000 ji wwf sooo G12C Detected amf 3000 2000 f 4000 0 32 34 36 38 E ra 44 4 4 50 52 54 f 6000 inl G12D Detected an 3000 2000 00 2 M g 3 a 2 u 6 amp 50 52 54 f ew Peak 6 sooo G12A Detected GCT an 3000 2000 f 1000 17 Example results of clinical FFPE samples for codon 13 analysis 34 3 35 37 3 UA Oa HS FDOT CFB KP Peak 4 G13C Detected TGT 43303508 338 OKO O DOE OE SOE DEY 9 BTS BHF TMP Y t G13D Detected 20007 Peak 2 4 GAT F Troubleshooting F 1 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 color 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 mu
9. de Change 1 Black G12V GGT gt GTT 2 Red G12S GGT gt AGT 3 Blue G12R GGT gt CGT 4 Black G12C GGT gt TGT 5 Red G12D GGT gt GAT 6 Blue G12A GGT gt GCT 7 Black G12V GGT gt GTT 8 Black Wild Type GGT Any peaks that do not match the peaks in this panel are to be disregarded Two GTT mutation peaks 1 and 7 are shown in this panel Results for NRAS Controls Codon 13 7 peaks will be presented as following 34 36 33 A a SHC 534 56 58 60 62 64 66 68 70 72 74 7 7 FI ttt ttt 5000 3 12 Peak Color Mutation Nucleotide Change 1 Black G13V GGT gt GTT 2 Red G13D GGT gt GAT 3 Blue G13A GGT gt GCT 4 Black G13C GGT gt TGT 5 Red G13S GGT gt AGT 6 Blue G13R GGT gt CGT 7 Black Wild Type GGT Any peaks that do not match the peaks in this panel are to be disregarded The pattern size or position of the peaks may vary slightly depending on instrument polymer type and the length of capillary Customer should validate the correct size for each peak by using the NRAS Controls Codon 12 and 13 Sample Analysis The wild type peak is a black peak on the right The mutation s will show as additional peak s The peak size and color of the mutation peaks in the NRAS Controls Codon 12 and 13 are used as references to identify mutations in the sample Any peaks that do not match the correct size and color are not considere
10. eptable for performing the assay Sequencer Applied Biosystems Genetic Analyzer Instrument Data Collection Genetic analyzer 3100 Data Collection Genetic analyzer 3700 Peay Genetic analyzer 3130 3500 Data Collection Genetic analyzer 3500 Software v1 0 Data Analysis GeneMapper Software 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 adjust the final concentration of extracted DNA to 20 80 ng pl When using TrimGen s WaxF ree DNA kit follow the user manual to perform PCR reaction Sequencer setup First time users should set up the analysis program for the ABI sequencer one time setup Af
11. not show the graphic data Diluting the final ST product with de ionized water and reloading the sample will easily resolve this problem The size standard may be miscalculated Check the size standard and manually correct the size standard see the sequencer s 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 all samples 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 Normally 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 support for further analysis In some circumstances when the sample DNA concentration is too low or the PCR did not amplify DNA properly 19 F 7 F 8 an unusual peak will appear in a very different position most of them are far from the wild type peak Any peaks outside of the data interpretation zone 25 80 on x
12. tation peaks have different peak size leak through will not affect data analysis F 2 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 18 F 3 F 4 F 5 F 6 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 automatically show 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 the size standard is too low the software cannot detect the size standard correctly and the program will
13. ter 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 pdf Step Ill Data Analysis Using GeneMapper www trimgen com docs Partlll Data Analysis GeneMapper pdf Important Spectral calibration is required before running the test The sequencer needs to be calibrated with the DS 32 calibration kit Applied Biosystems cat No 4345831 This is a one time calibration to set up spectral channels to collect the test results Refer to the DS 32 Matrix standards kit to prepare the DS 32 matrix standards Run a 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 45 sec 60 C 20 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 each time A 1 Prepare PCR Reaction Mix Master Mix 18 x 2 x 1 1 of Samples NR
14. tion 25 40 min Time varies depending on the type of sequencer Materials Provided The Mutector NRAS Mutation Detection kit contains reagents enough for 32 tests Reagents Quantity Description Master Mix 650 ul Master Mix Reagents for DNA amplification PCR primer mix for amplification of NRAS gene NRAS PCR Primers 50 ul codon12 and condon 13 C UP1 40 ul Enzyme 1 for cleanup of PCR products C UP2 40 ul Enzyme 2 for cleanup of PCR products C UP Buffer 430 ul Buffer for C UP reaction Pre mixed STA reagents for detection of NRAS NRAS ST 12 430 ul codon 12 mutations Pre mixed STA reagents for detection of NRAS NRAS ST 13 430 pi codon 13 mutations Pre mixed detection primers for codon 12 NRAS DP 12 80 ul mutations a Pre mixed detection primers for codon 13 NRAS DP 13 80 ul mutations NRAS CTL 12 120 ul Mutation controls for codon 12 NRAS CTL 13 120 ul Mutation controls for codon 13 Loading Buffer 1000 yl x 2 Sample loading buffer with size standards Light Sensitive Keep these reagents protected from direct light Materials required 0 2 ml PCR tubes 8 well strip tube DS 32 Matrix Standard kit Applied Biosystems Cat No 4345831 This kit is a one time calibration to set up 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 block is acc
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