myT BRAF Protocol - Swift Biosciences
Contents
1. Experiment Menu Experiment BRAF Typo Standard Curve Reagents TaqMan Reagents ECCIN Q9 Experiment Properties g Enter an experiment name select the instrument type select the type of experiment to set up then select materials and methods for the PCR reactions and instrument run How do you want to identify this experiment ExpenmentName BRAF Barcode Optional User Name Op amp onal Comments Optional Which instrument are you using to run the experiment PZA C I a S a Set up run and analyze an experiment using a 4 or 5 color 96 well system What type of experiment do you want to set up TET TERT a a a a a a Melt Curve genotyping Presence Absence l Use standards to determine the absolute quantity of target nucleic acid sequence in samples Which reagents do you want to use to detect the target sequence The PCR reactions contain primers designed lo ampaty te target sequence and a TaqMan probe designed to detect amplification of fe target sequence Which ramp speed do you want to use in the instrument run For optimal results with the standard ramp speed Applied Biosystems recommends using standard reagents for your PCR reactions Setup Plate Setup M Define Targets and Samples Define Targets Target Name Reporter Quencher Color BRAF Experiment Properties d Run Method Reaction Se2. aaa Primers myT BRAF qPCR Primers for Detection of Human BRAF V6OOE K For Research Use Only Swift Biosciences Inc All rights reserved Version 1112 Notice to Purchaser Limited License This product is for research use only and is licensed to the user under Swift Biosciences intellectual property only for the purchaser s internal research Purchase of this product does not convey to the purchaser any license to perform the Polymerase Chain Reaction PCR process under any third party rights PCR probes can be purchased from a variety of vendors including Applied Biosystems Life Technologies Roche Molecular Systems Inc F Hoffman La Roche Ltd Integrated DNA Technologies Biosearch Technologies Nanogen Inc and others The use of certain probes including TagMan MGB FAM TAMRA FAM BHQ VIC MGB in connection with PCR process may require a license from one or more of these vendors Please contact individual vendors for the necessity of obtaining licenses The purchase of myT BRAF or any other items delivered by Swift Biosciences hereunder does not either expressly or by implication provide a license to use any proprietary technology of these vendors Trademarks Used in this Manual myT is a trademark of Swift Biosciences Inc Maxima Probe qPCR Master Mix is a registered trademark and product of Fermentas now part of ThermoFisher TaqMan is a registered trademark of Roche Prime Time qPCR Probes is a registered
3. amplifiable copies are analyzed a 1 sensitivity limit which represents 10 mutant copies in 10 wild type copies can be achieved If only 10 amplifiable copies are analyzed a reduced 10 sensitivity limit can be achieved which represents 10 mutant copies in 10 wild type copies The cut off Ct value for detection of 10 mutant copies for the three thermocyclers tested are in the table below Thermocycler 10 Copy Ct Cut off Eo UR ae ABI 7500 gt 47 LightCycler 480 CFX96 If a Ct value is obtained that exceeds the cut off it is scored as negative or below the limit of detection for this assay Occasionally when amplifiable copy number is limiting a Ct value near the cut off will be obtained In this case the assay can be repeated to confirm a positive amplification signal If positive the Allele specific Ct value will be dependent on the percent tumor cell content and the tumor heterogeneity of the sample from which the DNA was derived Low percentage tumor cell samples will have limited sensitivity 13 Appendix Roche LightCycler 480 Run protocol 1 Turnon the LightCycler 2 Open LightCycler software 3 Click on New Experiment from Template 4 Select Monocolor Hydrolysis Probe UPL Probe Setup 1 Detection format Monocolor Hydrolysis Probe UPL Probe 2 Click on Customize select Dynamic and FAM 465 510 3 Block Size 96 4 Reaction Volume 25ul 5 Set Programs and Temperature target
4. a Ct of 26 7 is obtained 28 7 26 7 2 Ct so dilute sample 4 fold 3 If samples have a Ct value greater than the control DNA well add up to 10 amplifiable copies per 5 ul assuming that a two fold increase in DNA will decrease the Ct value by 1 Do not exceed 20 DNA per reaction volume as PCR inhibitors are present in FFPE preparations ABI 7500 Example If a Ct of 29 3 is obtained 29 3 27 3 2 so add 4 fold more DNA if possible 4 If samples have insufficient amplifiable copy number 1 sensitivity is not likely to be achieved as 1 represents 10 mutant copies in 10 total copies Based on Poisson distribution copy number less than 10 is not detected at 100 frequency in a single well reaction 5 If the Locus specific Ct value is gt 35 the amplifiable copy number is too low to proceed 6 Regarding the NTC either no amplification or an occasional Ct gt 38 may be obtained If the NTC or DNA Standard positive control fails contact technical service 11 Step 2 Allele specific BRAF V600E K qPCR Thaw reagents completely at room temperature Once thawed invert repeatedly or gently vortex and briefly centrifuge to collect contents To avoid cross contamination always briefly centrifuge DNA Standard and DNA samples prior to opening caps Also gently mix reactions containing 2X Master Mix to avoid formation of bubbles that can interfere with fluorescence detection Each reaction contains BRAF Allel
5. can Mode SYBR FAM only Assign Select Fluorophores FAM Assign your Sample Type for each well Unknown NTC Positive control Select Load FAM Click OK and save as template 19 EEEE 9 Select Next gt gt Run Setup Start Run Open the CFX96 lid Insert your plate in the CFX96 Close the lid Select Start run Save your experiment Wood Ie E
6. cific and Allele specific myT Primer stocks The final volume for each stock will be 198 72 270 ul Y Y VV WV Probes are light sensitive Avoid prolonged exposure to light once probe has been added 96 well plates are not supplied but the following have been tested using myT BRAF gt ABI 7500 Order from Applied Biosystems Life Technologies o 96 well optical reaction plates cat no 4306737 o MicroAmp optical Adhesive Film cat no 4311971 gt LightCycler 480 Order from Roche Applied Science o LightCycler 480 Multiwell Plate 96 white with sealing foils cat no 04729692001 gt CFX96 Order from Bio Rad o Multi plate Low profile unskirted 96 well plates natural cat no MLL 9601 o Microseal Film B cat no MSB1001 Notes Regarding DNA Samples gt For high quality DNA derived from cell lines or fresh frozen clinical samples UV absorbance readings correlate well with amplifiable content gt For DNA derived from formalin fixed paraffin embedded samples FFPE UV absorbance readings determine the DNA concentration but do NOT accurately determine amplifiable content due to DNA damage from fixation gt It is recommended to obtain UV absorbance readings for each sample in order to determine the amount of DNA to use in the Locus specific qPCR Step 1 gt Itis recommended to use 5 ng of high quality DNA or a range of 10 50 ng of FFPE DNA for the Locus specific qPCR gt Inthe case of heavily damaged samples
7. ck on START RUN in the upper right corner of the screen 18 Bio Rad CFX96 Run Protocol 1 2 3 Turn on CFX96 Open CFX96 software on your computer Select Create a new Run CFX96 Run Setup Protocol 1 2 3 4 Select Create New Sample Volume 25yl Edit protocol as followed Step 1 95 C 10 minutes 1 cycle Step 2 95 C 14 seconds Step 3 62 C 1 minute read Step 2 and 3 are repeated 45 or 60 cycles Click on OK and save protocol as a template BR Editor New E T File Settings Tools amp Insert Step Sample Volume pl Est Run Time 02 19 00 45 cycles of steps 2 and 3 are required for Locus specific reactions and 60 cycles for q Allele specific reactions 4 inser Step 2 950 C for0 14 3 620 C for 100 DE Insert Gradient Plate Read 4 GOTO2 60 more times END inset GOTO E insert Met Curve E Add Plate Read to Step SSS Zn step Options nv Delete Step 45 cycles of steps 2 and 3 are required for Locus specific reactions and 60 cycles for Allele specific reactions 5 Review your protocol setup and Select Next gt gt Run Setup Plate 1 EB LE d Click on Create new Select the wells containing a reaction on the grid Click on Settings Select Plate Type we suggest to use BR clear plates Select Plate Size 96 wells Select S
8. e specific Primers Probe 2X qPCR Master Mix 12 5 ul p mem oM om m DNA Template Total Volume 254 Remember to add 72 ul of resuspended probe to the myT Primer tube before initial use Make a cocktail with BRAF Allele specific Primers and 2X qPCR Master Mix in the amount needed for the number of reactions to be run plus up to 5 extra volume to compensate for pipetting loss maximum 28 samples plus 2 control wells Invert cocktail repeatedly to mix reagents and briefly centrifuge to collect contents Dispense 20 ul cocktail into each reaction well Add 5 ul sample DNA that corresponds to 10 amplifiable copies as determined from the Locus specific qPCR in Step 1 above Include a no template control NTC by adding 5 ul Nuclease free Buffer to one reaction well Include a 10 copy positive control by adding 5 ul DNA Standard to one reaction well Seal plate and briefly centrifuge 1000 2000 RPM for 15 seconds to collect contents Load plate into the selected thermocycler and follow run instructions for details see Appendix for ABI 7500 Roche LightCycler 480 or Bio Rad CFX96 instructions Cycling Temperature Cycling Time 5e c with FAM read disable any reads for passive reference dyes such as ROX 12 Determination of BRAF V600E K status for each sample For the Allele specific myT BRAF qPCR either a positive or negative amplification signal will be obtained If 10
9. ecessary use Nuclease free Buffer provided to dilute samples Include a no template control NTC by adding 5 ul Nuclease free Buffer to one reaction well Include a 10 copy positive control by adding 5 ul BRAF DNA Standard to one reaction well Seal plate and briefly centrifuge at 1000 2000 RPM for 15 seconds to collect contents WM OM m oa Load plate into the selected thermocycler and follow run instructions for details see Appendix for ABI 7500 Roche LightCycler 480 or Bio Rad CFX96 instructions Cycling Temperature Cycling Time 5 C m J with FAM read disable any reads for passive reference dyes such as ROX 10 Determination of amplifiable copy number for the allele specific assay 1 The control DNA Standard has 10 amplifiable BRAF copies per 5 ul and should have a Ct value as specified in the table below depending on the thermocycler used 10 is the recommended amplifiable copy number to place in the Allele specific assay Limiting the assay to 10 amplifiable copies reduces the likelihood of PCR inhibition and detection of low level cross contamination that can be present in FFPE samples Expected Average Ct value for Locus specific 10 copies ABI 7500 2 3 oo N LC 480 CFX96 2 If samples have a Ct value less than the control DNA well dilute with Nuclease free Buffer to 10 amplifiable copies per 5 ul assuming that a 2 fold dilution will increase the Ct value by 1 LC 480 Example If
10. gt 50 ng DNA can be placed into a reaction but inhibition of PCR is likely to occur Similarly it is not recommended to place greater than 20 volume of DNA per 25 ul reaction as PCR inhibitors are present in FFPE samples gt This assay has been tested using DNA isolated by the Qiagen QIAamp DNA FFPE Tissue Kit with RNase treatment not included in this kit Since RNA co purifies with DNA RNase treatment provides more accurate DNA quantification based on UV absorbance reading gt To avoid cross contamination that could lead to false positive results e Change gloves frequently e Use aerosol resistant pipette tips e Use pipettes dedicated for template and non template containing reagents e Maintain separate work areas for template and non template containing reagents e Routinely decontaminate work areas with 10 bleach and or UV light e Never open PCR reaction wells that resulted in allele specific amplification myT BRAF Workflow Isolate genomic DNA from samples 4 Obtain UV absorbance readings 4 Perform Locus specific qPCR Md Determine amplifiable copy number from Ct values H Perform Allele specific qPCR 4 Determine BRAF V600E K status The contents provided are sufficient to perform 60 reactions consisting of 30 Locus specific and 30 Allele specific reactions This enables testing of up to 28 samples when including a positive control and NTC if performed as a single qPCR run If testing is split int
11. j 0 Standard 1 0 Negative Control nw Setup Run method 1 Select Tabular View 2 Reaction Volume Per Well 25l 3 Holding Stage 1 step 95 C 10 minutes ramp rate 100 4 Cycling Stage 2 steps 5 Number of Cycles 45 or 60 cycles 95 C 14 seconds ramp rate 100 62 C 1 minute ramp rate 100 collect data on hold 45 cycles of Cycling Stage are required for Locus specific reactions and 60 cycles for Allele specific reactions 17 Run Method a Review the reaction volume and the thermal profile for the default run method If needed edit the default run method or select a run method from the library zz Experiment Properties Graphical View Tabular View Plate Setup Reaction Volume Per Well 25 uL Expert Mode SeleciView Millers costes CollectData v Open Run Method Save Run Method Revertto Defaults M Number of Cycles 60 Fl 45 cycles of Cycling E Enable AutoDelta H saing cie BEN Stage are required for Locus specific reactions and 60 cycles for Allele specific reactions Reaction Setup 7 PEGLA Ramp Rate 96 100 0 A Temperature C 95 0 Time 10 00 AutoDelta Temp AutoDelta Time Collect Data on Ramp m i Collect Data on Hold j Step 1 Step 1 Step 2 6 Open the door on the ABI 7500 Insert your plate Close the door 7 Cli
12. lification This high confidence Yes No clarity is a feature that is exclusive to myT Primer reagents Protocol The myT BRAF package provides sufficient reagents to perform a total of 60 reactions to assess BRAF mutations V600E K using the ABI 7500 Roche LightCycler 480 or Bio Rad CFX96 Real Time PCR Systems Mutation detection with myT BRAF consists of two steps 1 Locus specific qPCR gt A noncallele specific GPCR is performed to assess total mutant wild type amplifiable BRAF for each sample gt This determines the quantity of DNA to be used in step 2 for each sample gt Reagents for 30 reactions including controls are included 2 Allele specific qPCR gt A mutant allele specific BRAF qPCR is then performed to assess presence of V600E or V600K gt This assay DOES distinguish between wild type and mutant but DOES NOT distinguish between the two mutant alleles Results are reported as positive or negative for mutant BRAF for each sample with a sensitivity limit of 1 10 mutant copies in 10 wild type copies gt Reagents for 30 reactions including controls are included Reagents Included Reagent Mixes BRAF Locus specific Primers 198 ul Non allele specific myT BRAF primers BRAF Allele specific Primers 198 ul V600E K Allele specific myT BRAF primers Nuclease free Buffer For DNA sample dilution and NTC reactions NTC no template control Shipped in a separate box BRAF DNA Standard BRAF V600E c
13. o multiple batches total samples tested will be less since a positive control and NTC are required for each run For example if testing is batched into 3 qPCR runs the total number of samples analyzed will be 24 8 per run where 3 positive control and 3 NTC reactions are also run A 10 excess volume is included to compensate for pipetting loss Step 1 Locus specific BRAF qPCR Thaw reagents completely at room temperature Once thawed invert repeatedly or gently vortex and briefly centrifuge to collect contents To avoid cross contamination always briefly centrifuge DNA Standard and DNA samples prior to opening caps Also gently mix reactions containing 2X qPCR Master Mix to avoid formation of bubbles that can interfere with fluorescence detection Each reaction contains BRAF Locus specific Primers Probe 2X qPCR Master Mix DNA Template Total Volume 25W Remember to add 72 ul of resuspended probe to the myT Primer tube before initial use 1 Make a cocktail with BRAF Locus specific primers and 2X qPCR Master Mix in the amount needed for the number of reactions to be run plus up to 5 extra volume to compensate for pipetting loss maximum 28 samples plus 2 control wells 2 Invert tube with the cocktail repeatedly to mix reagents and briefly centrifuge to collect contents 3 Dispense 20 ul cocktail into each reaction well 4 Add 5 ul sample DNA corresponding to 5 ng high quality DNA or 10 to 50 ng of FFPE DNA If n
14. ontrol DNA 200 copies ul gt Store all reagents at 20 C upon arrival To avoid cross contamination store the myT Primers box separately from the DNA Standard box gt Refreeze unused myT Primers and DNA Standard at 20 C gt For best performance limit freeze thaw cycles to 4 es NEEDED RE oon SEE EE EII NIENNNNENM Reagents not included Reagents Scale Recommended Vendor qPCR Master Mix 2X 200 reactions Maxima Probe qPCR Master Mix 2X ThermoFisher Fermentas catalog K0261 Dual Labeled Probe Mini 0 5 nmol IDT PrimeTime Dual Labeled Probe Free of charge voucher provided Note myT BRAF has been optimized for use with the above reagents Reagents from other vendors may be substituted but substitutions may result in reduced assay performance or require the user to modify assay conditions to achieve maximal performance Probe sequence 5 56 FAM CAC CTC AGA TAT ATT TCT TCA TGA AGA CCT CAC AG 3IaBkFQ 3 When ordering this probe please include an internal Zen quencher gt Details on how to redeem the free of charge voucher for the dual labeled probe from IDT were sent with your order acknowledgement If you have any questions please contact Swift Technical Support at 734 330 2568 or technicalsupport swiftbiosci com Instructions for re suspension of probe Spin lyophilized probe to collect contents Resuspend in 167 ul of Nuclease free Buffer provided Distribute 72 ul each to the Locus spe
15. s as follows A Pre incubation Programs Program Name Cycles Analysis Mode Pre incubation 1 None Temperature Targets Target 9C Acquisition Mode Hold hh mm ss Ramp Rate 9C s 959C None 00 10 00 4 4 B Amplification Programs Program Name Cycles Analysis Mode Amplification 45 or 60 Quantification Temperature Targets Target 9C Acquisition Mode Hold hh mm ss Ramp Rate 9C s 95 None 00 00 14 4 4 62 Single 00 01 00 2 2 45 cycles of amplification are required for Locus specific reactions and 60 cycles for Allele specific reactions C Cooling Programs Program Name Cycles Analysis Mode Cooling 1 None Temperature Targets Target 9C Acquisition Mode Hold hh mm ss Ramp Rate 9C s 40 None 00 00 30 2 2 For all Temperature Targets Acquisition per C not used Sec Target 9C Step Size C Step Delay cycles 0 pre incubation c amplification cooing re hB Es His O e Vb OO em TS 6 Open the door insert your plate and close the door 7 Click on Start Run at the bottom right corner of the screen Life Technologies ABI 7500 Run protocol 1 Turn on the ABI 7500 2 Open ABI7500 software on your computer 3 Select Advanced Setup Setup Experiment properties 1 2 Name your experiment Select 7500 96 Wells Quantitation Standard Curve TaqMan Reagents Standard 2 hours to complete a run
16. trademark and product of Integrated DNA Technologies QIAamp is a registered trademark and product of Qiagen GmbH ABI 7500 Real Time PCR System is a trademark and product of Applied Biosystems now part of Life Technologies Corp CFX96 Real Time PCR Detection System is a trademark and product of Bio Rad Laboratories Inc LightCycler 480 Real Time PCR System is a registered trademark and product of Roche Applied Science myT Primer Technology myT Primers have unique structural and thermodynamic properties that make them highly sensitive to mismatch discrimination myT Primers are comprised of Primer and Fixer oligonucleotides with three functional domains the long Fixer domain provides a high level of specificity for genomic DNA templates the Primer domain is highly sensitive to single base mutations due to its very short length and the double stranded stem links the Fixer and Primer domains When a mutant specific myT Primer is combined with a reverse primer and hydrolysis probe myT Primers can detect 1 mutant BRAF V600E or K present in a background of 10 wild type genomic DNA copies without non specific amplification from wild type either a positive or negative amplification signal is generated and a delta Ct method to distinguish specific from non specific amplification is not required see data on page 4 Mutant Template Primer Fixer gt ee ae 3 Wild Type Template 5 3 Stem no extension Swift also offers m
17. tup FAM None your choice Experiment BRAF Type Standard Curve Assign Targets and Samples g instructions Define the targets to quantify and the samples to test in Ine reaction plate Target Name Reporter Quencher Color BRAF None 15 16 2 Define Targets and Samples Define Samples Name your samples Reagents TaqMan Reagents E 3 Assign Targets and Samples Select each well containing a reaction in View Plate Layout and assign Target BRAF Assign your particular samples the same way Select the dye to use as a passive reference None Define Targets and Sample Assign Targets and Samples n To set up standards Click Define and Set Up Standards Experiment Properties To set up unknowns Select wells assign target s select 1 Unknown as the task for each target assignment then assign a sample To set up negative controls Select wells assign target s ee N Negabve Control as the task for each target assignment i Assign target s to the selected wells View Well Table LETS Reaction Setup 2 3 E iw Matertals List Dew ew Mixed T Unknown EJ Standara Negative Control Assign sample s to the selected wells Sample 1 Assign sample s of selected well s to biological group Bolopcal Group meer p p Select the dye to use as the passive reference OCA enar Wells f 96 Unknown E
18. yT BRAF Ultra Primers for ultra low copy detection of V600E K from genomic DNA myT BRAF Ultra can detect a single mutant BRAF copy in 10 wild type BRAF copies Regardless of the sensitivity level of mutant BRAF detection needed myT Primer reagents offer increased specificity which provides higher confidence in test performance myT BRAF Performance Positive Negative 7 500 000 qe copies mutant n 4 ia 0 copy mutant 103 copies WT gDNA n 95 10 copies mutant n 4 E Mio copies mutant n 8 2 000 000 7 1 500 000 VA e f 1 000 000 j 500 000 f D A AZZ M M e EE a E I 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Cycle Cycle Positive amplification plot qPCR reactions containing mutant genomic DNA at the specified quantity in a background of 10 wild type genomic DNA resulted in BRAF V600E specific amplification 50 mutant content green n 4 replicates 10 mutant content yellow n 4 replicates and 1 mutant content blue n 8 replicates Assay performed on an ABI 7500 Negative amplification plot qPCR reactions containing 10 wild type genomic DNA no mutant DNA resulted in no amplification red in 95 separate reaction wells Assay performed on an ABI 7500 Conclusion These results demonstrate mismatch discrimination with very high specificity The result is clear and unambiguous eliminating the need for ACt analysis to distinguish specific from non specific amp
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