Type-it® HRM™ PCR Handbook
Contents
1. Figure 5 Programming the LightCycler 480 system for HRM analysis Note Times are entered in the format hh mm ss Program the cycling protocol described in Figure 4 For HRM analysis use 00 00 01 1 s at 65 C and 95 C Select Continuous for Acquisition Mode with 25 acquisitions per second resulting in a ramp rate of 0 02 C s Following HRM samples should be cooled after the last cycle by entering 00 00 01 1 s at 40 C not shown 20 Type it HRM PCR Handbook 07 2009 Protocol Analysis of SNPs by HRM This protocol is for use with the Rotor Gene Q Rotor Gene 6000 and LightCycler 480 All protocols for HRM analysis for the specific instruments and upcoming HRM instruments are available online at www qiagen com Products Type itHRMPCRKit aspx Important points before starting MH The optimized protocols in the handbook must be followed to ensure successful results E Always use a primer concentration of 0 7 pM E No optimization of the Mg concentration or the annealing temperature is required E Always start with the cycling conditions specified in this protocol E Optimal instrument and HRM analysis settings are a prerequisite for accurate genotyping results For details please refer to the manual provided with your HRM real time PCR instrument Procedure 1 Thaw 2x HRM PCR Master Mix primer solutions RNase free water template DNAs and control DNAs optional It is important to mix the solutions comp2. Type it HRM PCR Handbook For detection of gene mutations and SNPs by high resolution melting HRM analysis QIAGEN QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www qgiagen com Contents Kit Contents 4 Storage 4 Product Use Limitations 4 Product Warranty and Satisfaction Guarantee 5 Technical Assistance 5 Safety Information 6 Product Specifications 6 Quality Control 6 Introduction 7 HRM High Resolution Melting 7 General considerations for genotyping by HRM 9 Cyclers 11 Equipment and Reagents to Be Supplied by User 12 Important Notes 13 Protocols H Analysis of Gene Mutations and Microbial Genetic Differences by HRM 14 E Analysis of SNPs by HRM 21 Troubleshooting Guide 28 Appendix A HRM Instrument Setup and Data Analysis 30 Appendix B Starting Template 30 Appendix C Designing and Handling Primers 30 Ordering Information 33 Type it HRM PCR Handbook 07 2009 3 Kit Contents Type it HRM PCR Kit 10
3. 30s 55 C Extension 10s 72 C Activate single fluorescence data acquisition Suitable for PCR products up to 350 bp For PCR products gt 350 bp use s extension time per 25 bp of PCR product length Number of cycles 45 10 50 ng template DNA or 10 50 pg microbial DNA 50 1 9 ng template DNA or 1 9 pg microbial DNA HRM Analysis mode Melting curve is 05C 95 C Continuous fluorescence data acquisition Ramp rate 0 02 C s 25 acquisitions per second Cooling 1s 40 C Cooling samples after HRM Note Use maximum ramp rates for heating and cooling It is required to initially determine the melting point for each new HRM PCR product Run HRM analysis to span a temperature range from 65 C 95 C covering the full range of expected melting points In future experiments after determination of the Ta you may run HRM from 5 C below the lowest T of all expected PCR products to 5 C above the highest Ta of all PCR products in your experiment This may reduce the time required for HRM analysis 18 Type it HRM PCR Handbook 07 2009 Simp leProbe Mono Color Mydrolysis Probe UPL Probe Dual Color Hydrolysis Probe UPL Probe Multi Color Hydrolysis Probe Mono Color HybProbe Multi Color HybProbe Figure 3 Programming the LightCycler 480 for HRM analysis Choose detection format SYBR Green I HRM Dye Note If this channel for detection of EvaGreen dye is not chosen reduced fluorescence may be observed
4. 94545 Tel 510 265 1027 Fax 510 265 1352 Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the Type it HRM PCR Kit to the following terms 1 The Type it HRM PCR Kit may be used solely in accordance with the Type it HRM PCR Handbook and for use with components contained in the kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the Type it HRM PCR Handbook and additional protocols available at www qiagen com 2 Other than expressly stated licenses QIAGEN makes no warranty that this kit and or its use s do not infringe the rights of third parties 3 This kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated 5 The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components For updated license terms see www qi
5. Gene Q makes it the most precise and versatile realtime PCR cycler currently available Like the precursor model Rotor Gene 6000 each tube spins in a chamber of moving air keeping all samples at precisely the same temperature during rapid thermal cycling Detection is similarly uniform When each tube aligns with the detection optics the sample is illuminated and the fluorescent signal is rapidly collected from a single short optical pathway This thermal and optical uniformity results in sensitive precise and fast HRM analysis It also eliminates sample to sample variations and edge effects The comprehensive Rotor Gene Q software package supports all current state of the art HRM analysis procedures from basic to advanced algorithms Other real time cyclers with HRM abilities The LightCycler 480 as well as the Applied Biosystems 7500 Fast System and the Applied Biosystems 7900 System also enable HRM analysis with purchase of dedicated software The cyclers are block cyclers and have the advantage of working with standard PCR plate formats However during HRM analysis less reproducible and less precise high resolution melt values are obtained due to temperature gradients across the block and multiple complex optical pathways Type it HRM PCR Handbook 07 2009 11 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information c
6. We recommend using TE buffer 10 mM Tris Cl 1 mM EDTA pH 8 0 for standard primers Storage Primers should be stored in sterile nuclease free TE buffer in small aliquots at 20 C Standard primers are stable under these conditions for at least 1 year Dissolving primers Before opening a tube containing lyophilized primer centrifuge the tube briefly to collect all material at the bottom of the tube To dissolve the primer add the required volume of sterile nuclease free TE buffer mix and leave for 20 minutes to allow the primer to completely dissolve Mix again and determine the concentration by spectrophotometry as described below We do not recommend dissolving primers in water They are less stable in water than in TE buffer and some may not dissolve easily in water Concentration Spectrophotometric conversion for primers 1 Az unit 20 30 pg ml To check primer concentration the molar extinction coefficient E60 can be used A20 260 X molar concentration of primer or probe If the 2 value is not given on the data sheet supplied with the primers it can be calculated from the primer sequence using the following formula 20 0 89 x A x 15 480 C x 7340 G x 11 760 T x 8850 Type it HRM PCR Handbook 07 2009 31 Example Concentration of diluted primer 1 pM 1 x 10 M Primer length 24 nucleotides with 6 each of A C G and T bases Calculation of expected Aygo 0 89 x 6 x 15 480 6 x 7
7. for HRM analysis Choose detection format SYBR Green I HRM Dye Note If this channel for detection of EvaGreen dye is not chosen reduced fluorescence may be observed Detection Format fever Green I MRM Dye z Customize Block Size Plate 10 Reaction Volume 5 2 i ll A N Programs Program Name Cycles Analysis Mode Initial activation step 1 SiNone OR SNP cycling fas SjQuantitication Tr li SlHeiting Curves Okt fz 3 None gt n Al HRM SNP cycling Temperature Targets h Target Q Acquisition Mode Hold hh mm ss J Ramp Rate Cs Acquisitions per O i Step Size Q Step Delay cycles 95 Jes 00 00 10 sjo 0 k ss Sisingle 00 00 30 sjo sjo i AA 416 o Estimated Time bcm ss Figure 9 Programming the cycling protocol on the LightCycler 480 system for SNP analysis Note Times are entered in the format hh mm ss Important Set the initial activation step to 00 05 00 5 min at 95 C to activate HotStarTaq Plus DNA Polymerase not shown For the cycling steps use 00 00 10 10 s at 95 C and 00 00 30 30 s at 55 C Select Single for Acquisition Mode only for the 55 C step Adjust the number of cycles use 45 as a starting point Refer to Table 6 for details Use maximum heating cooling rates for all steps Programming of the HRM protocol is shown in Figure 10 Following HRM samples should be cooled after the last cycle by entering 00 00 01 1 s at 4
8. remove any liquid from the plate cover before PCR Follow mixing procedures in the protocol Decontaminate the realtime cycler according to the manufacturer s instructions Recalibrate the real time cycler according to the manufacturer s instructions Type it HRM PCR Handbook 07 2009 29 Appendix A HRM Instrument Setup and Data Analysis Please refer to your real time cycler user manual for correct instrument setup and data analysis If using the Rotor Gene Q refer to section 11 HRM data analysis of the Rotor Gene Q User Manual Note that in most cases the difference plot is required for accurate data analysis and genotyping results Melting peaks alone will not provide accurate results in many cases Appendix B Starting Template Sample degradation should be avoided during purification and storage Avoid excessive amounts of inhibitors from ethanol carryover To improve HRM results we recommend keeping the amount of template used consistent between samples Spectrophotometric analysis for determining DNA concentration and purity is recommended We recommend QIAGEN kits for sample preparation Note At 260 nm one absorbance unit is equal to 50 pg ml DNA Pure DNA will provide a 260 nm to 280 nm ratio of 1 8 Appendix C Designing and Handling Primers Sequence When designing primers for HRM PCR the following points should be noted MH Primers for HRM PCR should be 18 30 nucleotides in length E The melting
9. the green channel for the 72 C step Adjust the number of cycles use 40 as a starting point Refer to Table 2 for details Use maximum heating cooling rates for all steps Programming of the HRM protocol is shown in Figure 2 Click on a cycle below to modify it Hold Insert after Cyclin Insert before Remove Ramp from js 4 degrees to js degrees Rising by 0 1 degrees each step Wait for so seconds of pre melt conditioning on first step Wait for R seconds for each step afterwards Acquire to HAM on HAM Gain Optimisation I Optimise gain before melt on all tubes The gain giving the highest fluorescence less than 70 will be selected L Figure 2 Programming the Rotor Gene system for HRM analysis Program the cycling protocol for gene mutation analysis described in Figure 1 and Table 2 For HRM analysis ramp from 65 C to 95 C rising by 0 1 C each step Type it HRM PCR Handbook 07 2009 17 Q 5 z ro fe 5 n jo siskjpuy Table 3 Optimized cycling protocol for HRM analysis on the LightCycler 480 os Step Time Temp Additional comments c 5 2 Important Choose detection format id SYBR Green I HRM Dye 5 Initial PCR 5min 95 C HotStarTaq Plus DNA Polymerase is lt S activation step activated by this heating step 3 step cycling Important Optimal performance is only assured using these cycling conditions Denaturation 10s 95 C Annealing
10. 0 400 Catalog no 206542 206544 Number of 25 pl reactions 100 400 2x HRM PCR Master Mix containing 1x1l 3m 4x1 3 ml E HotStarTaq Plus DNA Polymerase E Type it HRM PCR Buffer with EvaGreen dye E QSolution E dNTP mix dATP dCTP dGTP dTTP RNase Free Water 1x2nl 2x2nml Handbook Storage The Type it HRM PCR Kit is shipped on dry ice The kit should be stored immediately upon receipt at 20 C in a constant temperature freezer and protected from light When stored under these conditions and handled correctly this product can be stored at least until the expiration date see the inside of the kit lid without showing any reduction in performance 2x HRM PCR Master Mix can be stored at 2 8 C for up to 2 months without showing any reduction in performance Product Use Limitations The Type it HRM PCR Kit is intended for molecular biology applications This product is neither intended for the diagnosis prevention or treatment of a disease nor has it been validated for such use either alone or in combination with other products Therefore the performance characteristics of the products for clinical use i e diagnostic prognostic therapeutic or blood banking are unknown All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable
11. 0 C not shown 26 Type it HRM PCR Handbook 07 2009 Detection Format ever creen 1 mem vye JOC BlockSize gt lt Plato D Reaction Volume 2 a _ Programe Name Initial activation step HRM SNP cycling Cooling 95 SdNS jo s s jouy Figure 10 Programming the LightCycler 480 system for HRM analysis Note Times are entered in the format hh mm ss Program the cycling protocol described in Figure 9 For HRM analysis use 00 00 01 1 s at 65 C and 95 C Select Continuous for Acquisition Mode with 25 acquisitions per second resulting in a ramp rate of 0 02 C s Following HRM samples should be cooled after the last cycle by entering 00 00 01 1 s at 40 C not shown Type it HRM PCR Handbook 07 2009 27 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Comments and suggestions No signal poor R value PCR or HRM or signal detected late in PCR a b c d e 28 Wrong cyc
12. 17 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 Spain Orders 91 630 7050 Fax 911 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN ees Sample amp Assay Technologies
13. 340 6 x 11 760 6 x 8850 x 1 x 10 0 232 The measured As should be within 30 of the theoretical value If the measured Az is very different to the theoretical value we recommend recalculating the concentration of the primers or having the primers resynthesized 32 Type it HRM PCR Handbook 07 2009 Ordering Information Product Contents Cat no Type it HRM PCR Kit 100 For 100 x 25 pl reactions 2x 206542 HRM PCR Master Mix containing HotStarlaq Plus DNA Polymerase EvaGreen dye Q Solution dNTPs and optimized concentration of MgCl and RNase free water Type it HRM PCR Kit 400 For 400 x 25 pl reactions 2x 206544 HRM PCR Master Mix containing HotStarlaq Plus DNA Polymerase EvaGreen dye Q Solution dNTPs and optimized concentration of MgCl and RNase free water Related products Type it Fast SNP Probe PCR Kit for 5 nuclease probe based SNP detection with reliably high call rates Type it Fast SNP Probe For 100 x 25 pl reactions 2x 206042 PCR Kit 100 Fast SNP Probe PCR Master Mix 5x Q Solution RNase Free Water Type it Mutation Detect PCR Kit for reliable detection of mutations by multiplex PCR Type it Mutation Detect For 70 x 25 yl reactions 206341 PCR Kit 70 Multiplex PCR Master Mix 5x Q Solution RNase Free Water and 10x Coralload Dye Type it Microsatellite PCR Kit for reliable microsatellite analysis by multiplex PCR Type it Microsatellite For 70 x 25 pl reac
14. 50 bp For PCR products gt 350 bp use s extension time per 25 bp of PCR product length Number of cycles 40 10 50 ng template DNA or 10 50 pg microbial DNA A5 1 9 ng template DNA or 1 9 pg microbial DNA HRM 2s 65 95 C Fluorescence data acquisition One for details see page 10 increments It is required to initially determine the melting point for each new HRM PCR product Run HRM analysis to span a temperature range from 65 C 95 C covering the full range of expected melting points In future experiments after determination of the Ta you may run HRM from 5 C below the lowest T of all expected PCR products to 5 C above the highest T of all PCR products in your experiment This may reduce the time required for HRM analysis 16 Type it HRM PCR Handbook 07 2009 Click on a cycle below to modify it Hold Insert after re eset ate Remove This cycle repeats 40 timers Click on one of the steps below to modify it or press or to add and remove steps for this cycle 2 55 deg for 30 secs Timed Step 72 deg 10 seconds Acquiring to Cycling A 95 deg for 10 secs Figure 1 Programming the cycling protocol on the Rotor Gene system for gene mutation analysis Important Set the hold step to 5 min at 95 C to activate HotStarTaq Plus DNA Polymerase not shown For the cycling steps use 95 C for 10 s 55 C for 30 s and 72 C for10 s with data acquiring to Cycling A on
15. A Polymerase to eliminate nonspecific amplification products and provide reliable results Q Solution included in the master mix ensures specific amplification of difficult genomic loci leading to successful results The Type it HRM PCR Kit facilitates multiple applications including SNP genotyping Mutation discovery Scanning for mutations in disease and cancer related genes Identification of candidate predisposition genes Genetic association studies DNA fingerprinting Species identification and genotyping For DNA methylation analysis by HRM use the EpiTect HRM PCR Kit HRM High Resolution Melting High resolution melting analysis is an innovative technique that is based on analysis of DNA melting HRM characterizes DNA samples according to their dissociation behavior as they transition from double stranded DNA dsDNA to single stranded DNA ssDNA with increasing temperature Before performing HRM analysis the target sequence must be amplified to a high copy number in the presence of the dsDNA binding fluorescent dye EvaGreen The dye does not interact with ssDNA but actively binds to dsDNA and fluoresces brightly when bound Change in fluorescence can be used to measure the increase in DNA concentration during PCR and then to directly measure thermally induced DNA melting by HRM To perform high resolution melting analysis the temperature is increased from a lower to a higher temperature The fluorescence of EvaGreen is me
16. BIOTIUM INC and QIAGEN and the manufacture use sale or import of this product is subject to one or more of U S Patent Nos application Nos and corresponding international equivalents owned by BIOTIUM and ALLELOGIC The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer where such research does not include testing analysis or screening services for any third party in return for compensation on a per test basis The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For information on purchasing a license to this product for purposes other than research contact BIOTIUM INC Business Development 3423 Investment Blvd Suite 8 Hayward CA
17. Ensure that all reagents buffers and solutions used for isolating and dilution of template nucleic acids are free of nucleases RNases DNases Type it HRM PCR Handbook 07 2009 Comments and suggestions g Insufficient or degraded template DNA Check if template amount and PCR cycle number were used as specified in the protocol Tables 1 6 Increase the amount of template DNA if possible Increased fluorescence or C value for No Template control a Contamination of reagents b Contamination of real time cycler Discard all the components of the HRM PCR assay e g master mix primers Repeat the assay using new components and decontaminated pipettes and consumables Decontaminate the realtime cycler according to the manufacturer s instructions Variability in signal C and or R in HRM between replicates or samples a Problem with template DNA b Bubbles in the wells c Reaction components improperly mixed d Contamination of real time cycler e Real time cycler no longer calibrated Recheck the DNA concentrations of the samples Ensure that comparable amounts of DNA are used in all samples noting that all samples should not differ by more than three C values Use a different HRM genotyping assay to check the integrity of the genomic DNA in all samples Note that rare or new genetic variants may generate results outside of the expected ranges Spin down plates to remove air bubbles and
18. PCR product length Number of cycles AO 10 50 ng template DNA or 10 50 pg microbial DNA A5 1 9 ng template DNA or 1 9 pg microbial DNA HRM 2s 65 95 C Fluorescence data acquisition 0 1 C increments SdNS jO sisAjouy reduce the time required for HRM analysis Type it HRM PCR Handbook 07 2009 It is required to initially determine the melting point for each new HRM PCR product Run HRM analysis to span a temperature range from 65 C 95 C covering the full range of expected melting points In future experiments after determination of the Ta you may run HRM from 5 C below the lowest T of all expected PCR products to 5 C above the highest T of all PCR products in your experiment This may 23 a Z N fa a ie i lt Click on a cycle below to modify it Hold Insert after HRM Insert before Remove This cycle repeats 40 time s Click on one of the steps below to modify it or press or to add and remave steps for this cycle Timed Step 55 deg 30 seconds Acquiring to Cycling A on Green I Long Range I Touchdown 95 deg for 10 secs 55 deg for 30 secs Figure 6 Programming the cycling protocol on the Rotor Gene system for SNP analysis Important Set the hold step to 5 min at 95 C to activate HotStarTaq Plus DNA Polymerase not shown For the cycling steps use 95 C for 10 s and 55 C for 30 s with data acquiring to Cycling A o
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20. asured continuously as the temperature is increased and is plotted against the temperature EvaGreen fluoresces as long as it is bound to dsDNA Due to the amplification procedure before the HRM analysis fluorescence will be high at the beginning of the HRM analysis Upon melting of dsDNA EvaGreen is released and the fluorescence is reduced to a background level Type it HRM PCR Handbook 07 2009 7 HRM is easier and more cost effective than probe based genotyping assays and unlike conventional methods it is a closed tube system that prevents contamination with PCR products 2x HRM PCR Master Mix 2x HRM PCR Master Mix ensures highly specific amplification as well as flexible rapid and sensitive analysis of gene mutations and SNPs and enables genotyping via high resolution melting The components of 2x HRM PCR Master Mix include HotStarlaq Plus DNA Polymerase Type it HRM PCR Buffer Q Solution and dNTPs The optimized master mix ensures that the PCR products are amplified with high specificity and efficiency for successful HRM analysis even with difficult genomic loci HotStarTaq Plus DNA Polymerase HotStarTaq Plus DNA Polymerase is a modified form of QIAGEN Taq DNA Polymerase and is provided in an inactive state and has no enzymatic activity at ambient temperature This prevents the formation of misprimed products and primer dimers during reaction setup and the first denaturation step Competition for reactants by PCR artifact
21. below 30 and differing by no more than 3 C values E Itis recommended to use control DNA and sample DNA of comparable integrity For example for analyzing samples from FFPE embedded tissues control DNA should also be derived from FFPE tissues with comparable integrity E DNA samples used for HRM should be normalized in concentration All DNA samples should be quantified and then adjusted to the same concentration using the same dilution buffer Type it HRM PCR Handbook 07 2009 9 MH Use sufficient PCR cycles so that all samples have reached the plateau phase of PCR to ensure that comparable amounts of PCR product are generated Note that the amount of DNA affects the melting temperature of the PCR product Check the protocols for details Assay design Design assays with PCR product lengths of 70 350 bp For SNP analysis use of PCR products of 70 150 bp is recommended Larger products can be analyzed successfully but usually provide lower resolution This is because for example a single base variation has a greater effect on the melting behavior of for example a 100 bp PCR product than on a 350 bp PCR product Primers E Design primers allowing specific amplification Perform a BLAST search to ensure specific primer binding See Appendix C page 30 for details E The melting temperature of primers used for PCR with subsequent HRM analysis should be at least 56 C The melting temperature of primers can be calculated using th
22. ction Total volume per 25 pl 10 pl reaction If your realtime cycler requires a final reaction volume other than 25 pl adjust the amount of master mix and all other reaction components accordingly t A 10 pM primer mix consists of 10 pM forward primer and 10 pM reverse primer 5 Program the real time cycler according to Table 5 or 6 pages 23 25 Note Check the real time cycler s user manual for correct instrument setup 6 Place the PCR tubes or plate in the real time cycler and start the PCR cycling program followed by HRM analysis 7 Perform data analysis Before performing data analysis for real time PCR and HRM specify the analysis settings See the realtime PCR instrument manual for details Note Real time cyclers not listed in this handbook often do not have the ability to perform HRM analysis 22 Type it HRM PCR Handbook 07 2009 Table 5 Optimized cycling protocol for HRM analysis of SNPs on the Rotor Gene Q and Rotor Gene 6000 Step Time Temp Additional comments Initial PCR 5min 95 C HotStarlaq Plus DNA Polymerase is activation step activated by this heating step 2 step cycling Important Optimal performance is only assured using these cycling conditions Denaturation 10s 95 C Annealing Extension 30s 55 C Activate fluorescence data acquisition on the green channel Suitable for PCR products up to 200 bp For PCR products gt 200 bp use additional 1 s extension time per 25 bp of
23. e formula below T 2 C x number of A T 4 C x number of G C Mm Whenever possible design primer pairs with similar T values M Check the concentration and integrity of primers before starting Typically standard primer quality primers are sufficient for HRM For details see Appendix C page 30 E Important Always start with a primer concentration of 0 7 pM HRM analysis It is required to initially determine the melting point for each new HRM PCR product Run HRM analysis to span a temperature range from 65 95 C covering the full range of expected melting points In future experiments after determination of the Ta you may run HRM from 5 C below the lowest T of all expected PCR products to 5 C above the highest T of all PCR products in your experiment This may reduce the time required for HRM analysis Data analysis Check that the PCR result contains only specific product Samples showing postPCR artifacts such as primer dimers or nonspecific products can make HRM results difficult to interpret The Type it HRM PCR Kit ensures maximum specificity with minimal need for optimization 10 Type it HRM PCR Handbook 07 2009 Cyclers Rotor Gene Q and Rotor Gene 6000 QIAGEN s real time PCR cycler the Rotor Gene Q combines multiple optimized design features to provide outstanding performance and reliable results for demanding research applications The unique centrifugal rotary design of the Rotor
24. e solutions completely before use to avoid localized concentrations of salt 2 Prepare a reaction mix according to Table 1 page 15 It is recommended to prepare a volume of reaction mix 10 greater than that required for the total number of reactions to be performed Reaction setup can be done at room temperature 15 25 C However it is recommended to keep the individual reagents samples and controls on ice 3 Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tubes or the wells of a PCR plate 4 Add equal amounts and volumes of template DNA 1 50 ng genomic DNA or 1 50 pg microbial DNA same amount for each sample to the individual PCR tubes or wells and mix thoroughly Add sufficient DNA so that all samples show Cz values below 30 Samples should not differ by more than three C values 14 Type it HRM PCR Handbook 07 2009 Table 1 Reaction composition using 2x HRM PCR Master Mix reaction Volume per 10 pl Volume per reaction 25 pl 384 well Component reaction plate Final concentration Reaction mix 2x HRM PCR 12 5 pl 5 0 pl 1x Master Mix 10 pM primer mixt 1 75 pl 0 7 pl 0 7 pM forward primer 0 7 pM reverse primer RNase free water Variable Variable Template DNA Variable Variable Eukaryotic 1 50 ng added at step 4 equal volume equal volume DNA reaction for all for all Microbial 1 50 pg reactions reactions DNA reaction use equal amounts for each reaction Total vol
25. ese cycling conditions Activate single fluorescence data acquisition Suitable for PCR products up to 200 bp For PCR products gt 200 bp use additional 1 s extension time per 25 bp of PCR product length 10 50 ng template DNA or 10 50 pg microbial DNA 1 9 ng template DNA or 1 9 pg microbial DNA Analysis mode Melting curve Continuous fluorescence data acquisition Ramp rate 0 02 C s 25 acquisitions per second Cooling samples after HRM SdNS JO sisAjouy Note Use maximum ramp rates for heating and cooling lt is required to initially determine the melting point for each new HRM PCR product Run HRM analysis to span a temperature range from 65 C 95 C covering the full range of expected melting points In future experiments after determination of the Ta you may run HRM from 5 C below the lowest T of all expected PCR products to 5 C above the highest Ta of all PCR products in your experiment This may reduce the time required for HRM analysis Type it HRM PCR Handbook 07 2009 25 7 a Z 7e KA a ie lt Run Protocol J Data Run Hotes Simp leProbe Hono Color Hydrolysis Probe UPL Probe n n a a A Dual Color Hydrolysis Probe UPL Probe Programs Program Ni u1c1 Color Hydrolysis Probe Cycles Analysis Mode if Hono Color HybProbe b Program 1 y Multi Color MybProbe 4 i Figure 8 Programming the LightCycler 480
26. fied form of a DNA Polymerase recombinant 94 kDa DNA polymerase originally isolated from Thermus aquaticus cloned into E coli Deoxynucleoside triphosphate DNA deoxynucleotidyltransferase EC 2 7 7 7 The enzyme is activated by a 5 minute 95 C incubation step Type it HRM PCR Novel PCR buffer for highly specific amplification with Buffer subsequent high resolution melting analysis EvaGreen Novel dsDNA binding fluorescent dye allowing highly efficient and inhibition free PCR amplification and ideally suited for HRM analysis QSolution For successful amplification of difficult genomic loci Contained in the master mix at optimized concentration dNTP mix Contains dATP dCTP dGTP and dTTP of ultrapure quality RNase free water Ultrapure quality PCR grade Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of Type it HRM PCR Kit is tested against predetermined specifications to ensure consistent product quality 6 Type it HRM PCR Handbook 07 2009 Introduction The Type it HRM PCR Kit is provided in a convenient master mix format for detection of gene mutations or SNPs via high resolution melting analysis HRM HRM technology enables rapid characterization of DNA samples based on their melting behavior following PCR amplification The kit contains the novel double stranded DNA binding fluorescent dye EvaGreen and includes an optimized HRM buffer and HotStarTaq Plus DN
27. guidelines 4 Type it HRM PCR Handbook 07 2009 Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www giagen com Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If y
28. i Spin Columns 69504 Tissue Kit 50 Proteinase K Buffers Collection Tubes 2 ml The Type it Fast SNP Probe PCR Kit the Type it Mutation Detect PCR Kit and the Type it Microsatellite Kit are intended for research use No claim or representation is intend ed to provide information for the diagnosis prevention or treatment of a disease For up to date licensing information and productspecific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor Larger kit sizes available see www qiagen com t Contains 15 mM MgClo Contains 3 mM MgCl and 400 pM each dNTP 34 Type it HRM PCR Handbook 07 2009 Trademarks QIAGEN QlAamp Coralload DNeasy EpiTect HotStarlaq Type it Q Solution QIAGEN Group BLAST US National Library of Medicine EvaGreen Biotium HRM Rotor Gene Corbett Research Pty Ltd LightCycler Roche Group Applied Biosystems Applera Corporation SYBR Molecular Probes Inc Trizma Sigma Aldrich Co The purchase of this product includes a limited non transferable license under U S patent No 5 871 908 and all continuations and divisional and corresponding claims in patents and patent applications outside the United States owned by Roche Diagnostics GmbH for internal research use or for non in vitro diagnostics a
29. leaner SYR Green I HRN Dye Cee stock size Plate 10 Reaction Volume 3 95 None 00 00 10 4 4 None 00 00 30 2 2 o o so o B0 o o Target Q Acquisition Mode Hold hh mm ss Ramp Rate C s Acquisitions per C Step Delay cyclos 5 letnletoets 72 Single 00 00 10 44 g a M A HRI SNP cycling Temperature Targets M Figure 4 Programming the cycling protocol on the LightCycler 480 system for gene mutation analysis Note Times are entered in the format hh mm ss Important Set the initial activation step to 00 05 00 5 min at 95 C to activate HotStarlaq Plus DNA Polymerase not shown For the cycling steps use 00 00 10 10 s at 95 C 00 00 30 30 s at 55 C and 00 00 10 10s at 72 C Select Single for Acquisition Mode only for the 72 C step Adjust the number of cycles use 45 as a starting point Refer to Table 3 for details Use maximum heating cooling rates for all steps Programming of the HRM protocol is shown in Figure 5 Following HRM samples should be cooled after the last cycle by entering 00 00 01 1 s at 40 C not shown Type it HRM PCR Handbook 07 2009 19 e 5 ro fe 5 n jo siskjouy Analysis of Gene Mutations r E Pr i pock sie o sitet tg Curves Initial activation step 1 afio HRN gene mutations cycling 45 Quantification S Cooling 1 i
30. letely before use to avoid localized concentrations of salt 2 Prepare a reaction mix according to Table 4 page 22 It is recommended to prepare a volume of reaction mix 10 greater than that required for the total number of reactions to be performed Reaction setup can be done at room temperature 15 25 C However it is recommended to keep the individual reagents samples and controls on ice 3 Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tubes or the wells of a PCR plate 4 Add equal amounts and volumes of template DNA 1 50 ng genomic DNA or 1 50 pg microbial DNA same amount for each sample to the individual PCR tubes or wells and mix thoroughly Add sufficient DNA so that all samples show Cr values below 30 Samples should not differ by more than three Cy values Type it HRM PCR Handbook 07 2009 21 SdNS jO sisAjouy Table 4 Reaction composition using 2x HRM PCR Master Mix Volume per 10 pl Volume per reaction 25 pl 384 well Component reaction plate Final concentration A Reaction mix a Z 2x HRM PCR 12 5 pl 5 0 pl 1x N 5 Master Mix 2 10 pM primer mixt 1 75 pl 0 7 pl 0 7 pM forward primer 5 0 7 pM reverse primer RNase free water Variable Variable Template DNA Variable Variable Eukaryotic 1 50 ng added at step 4 equal volume equal volume DNA reaction for all for all Microbial 1 50 pg reactions reactions DNA reaction use equal amounts for each rea
31. ling conditions HotStarTaq Plus DNA Polymerase not activated Pipetting error or missing reagent Wrong or no detection step in real time analysis Poor PCR efficiency Problems with starting template DNA Always start with the optimized cycling conditions specified in the protocols Ensure that the cycling program includes the HotStarTaq Plus DNA Polymerase activation step 5 min at 95 C as described in the protocols Check the concentrations and storage conditions of the reagents including primers and template nucleic acid See Appendix C page 30 for details on evaluating the concentration of primers Repeat the assay For realtime analysis ensure that fluorescence detection takes place during the 72 C extension step for the gene mutation protocols or during the combined annealing extension step for the SNP protocol Use the primer concentrations given in the protocol See Appendix C page 30 for details on determining the concentration of primers Avoid repeated freezing and thawing of primers Prepare small aliquots and only thaw a few times Check the concentration storage conditions and quality of the template and control DNA If necessary make new serial dilutions of the template DNA from the stock solutions Repeat the assay using the new dilutions Efficient removal of PCR inhibitors is essential for optimal results Purify nucleic acids from your sample using an appropriate purification method
32. n the green channel for the 55 C step Adjust the number of cycles use 40 as a starting point Refer to Table 5 for details Use maximum heating cooling rates for all steps Programming of the HRM protocol is shown in Figure 7 NAIA Click on a cycle below to modify it Hold Insert after Cyclin D Insert before Remove Ramp from 65 degrees to 95 degrees Rising by 01 degree each step Wait for 30 seconds of pre melt conditioning on first step Waitfor 2 j seconds for each step afterwards Acquire to HAM A on HRM r Gain Optimisation V Optimise gain before melt on all tubes The gain giving the highest fluorescence less than 70 will be selected Figure 7 Programming the Rofor Gene system for HRM analysis Program the cycling protocol described in Figure 6 and Table 5 For HRM analysis ramp from 65 C to 95 C rising by 0 1 C each step 24 Type it HRM PCR Handbook 07 2009 Table 6 Optimized cycling protocol for HRM analysis of SNPs on the LightCycler 480 Step Time Temp Additional comments Initial PCR activation step 5min 95 C 2 step cycling 10s 30s 95 C 55 C Denaturation Annealing Extension Number of cycles 45 50 HRM ls 65 C 95 C ls 40 C Cooling Important Choose detection format SYBR Green I HRM Dye HotStarTaq Plus DNA Polymerase is activated by this heating step Important Optimal performance is only assured using th
33. onsult the appropriate material safety data sheets MSDSs available from the product supplier Primers The Type it HRM PCR Kit can be used with standard quality primers that can be purchased from established oligonucleotide manufacturers Lyophilized primers should be dissolved in TE buffer to provide a stock solution of 100 pM concentration should be checked by spectrophotometry Primer solutions should be stored in aliquots at 20 C Avoid repeated freeze thaw cycles of primers Prepare working solutions by dilution in TE e g each primer at 10 pM and store in small aliquots for single use Nuclease free DNase free consumables special care should be taken to avoid nuclease contamination of all reagents and consumables used to set up PCR Optical PCR tubes or plates use thin walled PCR tubes or plates recommended by the manufacturer of your real time cycler Optional Trizma base and EDTA for preparing TE buffer for storing primers see Appendix C page 30 Use RNase DNase free water and plastic consumables to prepare TE buffer Type it HRM PCR Handbook 07 2009 Important Notes For optimal results with the Type it HRM PCR Kit the optimized protocols in the handbook must be followed Following the protocols will ensure successful results without further optimization of PCR parameters such MgCl concentration or annealing temperature No template control NTC All detection experiments should include an NTC containing all
34. ou have any questions or experience any difficulties regarding the Type it HRM PCR Kit or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www giagen com Type it HRM PCR Handbook 07 2009 5 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 Product Specifications 2x HRM PCR Master Mix contains HotStarTaq Plus HotStarlaq Plus DNA Polymerase is a modi
35. pplication with authorized reagents with regard to Melting Curve Analysis No right is conveyed expressly by implication or estoppel under any other patent or patent claims owned by Roche Diagnostics GmbH or by any other Party The purchase price of this product includes a limited non transferable immunity from suit under U S Patents Nos 5 994 056 and 6 171 785 and corresponding patent claims outside the United States owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd Roche to use only this amount of the product for dsDNA binding dye processes covered by said patents solely for the purchaser s own internal research This product is also a Licensed dsDNA Dye Binding Kit for use with service sublicenses available from Applied Biosystems No right under any other patent claims such as apparatus or system claims in U S Patent No 6 814 934 and no right to use this product for any other purpose or for commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is hereby granted expressly by implication or by estoppel This product is for research use only Diagnostic uses 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 This product is provided under an agreement between
36. provided at www qiagen com Products Type it HRMPCRKit aspx Protocol Analysis of Gene Mutations and Microbial Genetic Differences is found on page 14 and is recommended for all HRM applications except SNP genotyping Protocol Analysis of SNPs by HRM is found on page 21 and is recommended for SNP genotyping by HRM General considerations for genotyping by HRM M Always check the realtime PCR instrument manual for details on HRM setup and analysis on your instrument E Note that conventional melting curve analysis as done for SYBR Green based detection and HRM are not the same HRM requires dedicated reaction chemistry as well as an HRM real time PCR instrument with dedicated heating algorithm and software Template E Purified genomic DNA of every origin suitable for PCR with respect to purity and concentration can be used to successfully perform HRM with the Type it HRM PCR Kit Mit is recommended to use the same genomic DNA purification procedure for all samples being analyzed by HRM This avoids introduction of variations due to differing compositions of elution buffers used in different extraction methods E To avoid any reduction in performance we recommend using QIAGEN genomic DNA purification kits such as QlAamp or DNeasy Kits E Use 1 ng to 50 ng of template genomic DNA or 1 50 pg microbial DNA per 25 pl reaction E Important Use comparable amounts of template genomic DNA for all samples resulting in Cy values
37. s is therefore avoided enabling high PCR specificity and accurate quantification The enzyme is activated at the start of a reaction by a 5 minute 95 C incubation step The hot start enables reactions to be set up rapidly and conveniently at room temperature EvaGreen EvaGreen is a novel fluorescent dye which selectively binds to dsDNA Upon binding fluorescence is strongly increased The spectral properties of EvaGreen are very similar to those of SYBR Green I The absorbance maximum is 500 nm with DNA bound and the emission maximum is 530 nm This allows easy detection of EvaGreen on channels filters preset for HRM analysis and SYBR Green detection In contrast to SYBR Green EvaGreen can be used in higher concentrations and shows equal binding affinity for GC rich and AT rich regions with no apparent sequence preference This makes EvaGreen an ideal dye for HRM analyses of all types of PCR products Protocol selection The Type it HRM PCR Kit has been optimized for use with the following real time cyclers Rotor Gene Q Rotor Gene 6000 and LightCycler 480 8 Type it HRM PCR Handbook 07 2009 This handbook contains 2 protocols for use with these cyclers The Applied Biosystems 7500 Fast System and Applied Biosystems 7900 can also be used with both optimized protocols in this handbook Note that both instruments essentially require a dedicated calibration for the HRM dye in use before the experiment A detailed protocol is
38. temperature of primers used for HRM PCR should be at least 56 C The melting temperature of primers can be calculated using the formula below Ta 2 C x number of A T 4 C x number of G C HM Whenever possible design primer pairs with similar T values M Primers for HRM PCR should have a GC content of 40 60 E Avoid complementarity of 2 or 3 bases at the 3 ends of primer pairs to reduce primer dimer formation Avoid mismatches between the 3 end of the primer and the target template sequence E Avoid runs of 3 or more G and or C at the 3 end E Avoid complementary sequences within primers and between primer pairs E Ensure primer sequence is unique for your template sequence Check similarity to other known sequences with BLAST www ncbi nlm nih gov blast Blast cgi or primer BLAST www ncbi nlm nih gov tools primer blast 30 Type it HRM PCR Handbook 07 2009 Commercially available computer software e g OLIGO 6 Rychlik 1999 or web based fools such as Primer3 Steve Rosen amp Helen Skaletsky 2000 frodo wi mit edu cgi bin primer3 primer3_www cgi can also be used for primer design Handling and storing primers Guidelines for handling and storing primers are given below For optimal results we recommend only combining primers of comparable quality Storage buffer Lyophilized primers should be dissolved in a small volume of low salt buffer to give a concentrated stock solution e g 100 pM
39. the components of the reaction except for the template This enables detection of potential contamination Positive control Include at least one gDNA control of known genotype for each assay tested in the experiment to be used as reference For a SNP genotyping experiment at least one control is needed for each possible genotype wild type heterozygote variant Type it HRM PCR Handbook 07 2009 13 Protocol Analysis of Gene Mutations and Microbial Genetic Differences by HRM This protocol is for use with the Rotor Gene Q Rotor Gene 6000 and LightCycler 480 All protocols for HRM analysis for the specific instruments and upcoming HRM instruments are available online at www giagen com Products Type itHRMPCRKit aspx Important points before starting N aos 5 2 2 B N gt 5 c o lt 55 The optimized protocols in the handbook must be followed to ensure successful results E Always use a primer concentration of 0 7 pM E No optimization of the Mg concentration or the annealing temperature is required E Always start with the cycling conditions specified in this protocol H Optimal instrument and HRM analysis settings are a prerequisite for accurate genotyping results For details please refer to the manual provided with your HRM real time PCR instrument Procedure 1 Thaw 2x HRM PCR Master Mix primer solutions RNase free water template DNAs and control DNAs optional It is important to mix th
40. tions 206241 PCR Kit 70 Multiplex PCR Master Mix 5x Q Solution and RNase Free Water Larger kit sizes available see www qiagen com Master mix contains HotStarlaq Plus DNA Polymerase ROX dye and dNTPs with optimized concentration of MgCl and Q Solution Master mix contains HotStarlaq Plus DNA Polymerase optimized MgCl concentration and 200 pM each dNTP a Type it HRM PCR Handbook 07 2009 33 Ordering Information Product Contents Cat no HotStarTaq Plus DNA Polymerase for highly specific hot start PCR without optimization HotStarTaq Plus DNA 250 units HotStarlaq Plus DNA 203603 Polymerase 250 U Polymerase 10x PCR Buffer 10x Coralload PCR Buffer 5x QSolution 25 mM MgCl HotStarTaq Plus Master Mix Kit for fast and highly specific amplification HotStarTaq Plus 3 x 0 85 ml HotStarTaq Plus Master 203643 Master Mix Kit 250 Mix containing 250 units of HotStarlaq Plus DNA Polymerase total 1 x 0 55 ml Coralload Concentrate 2 x 1 9 ml RNase Free Water for 250 x 20 pl reactions QlAamp DNA Kits for genomic mitochondrial bacterial parasite or viral DNA QlAamp DNA Mini For 50 DNA preps 50 QlAamp Mini 51304 Kit 50 Spin Columns QIAGEN Proteinase K Reagents Buffers Collection Tubes 2 ml DNeasy Blood amp Tissue Kits for purification of total DNA from animal blood and tissues and from cells yeast bacteria or viruses DNeasy Blood amp 50 DNeasy Min
41. ume per 25 pl 10 pl and all other reaction components accordingly If your realtime cycler requires a final reaction volume other than 25 pl adjust the amount of master mix t A 10 pM primer mix consists of 10 pM forward primer and 10 pM reverse primer 5 Program the real time cycler according to Table 2 or 3 pages 16 18 Note Check the real time cycler s user manual for correct instrument setup 6 Place the PCR tubes or plate in the real time cycler and start the PCR cycling program followed by HRM analysis 7 Perform data analysis Before performing data analysis for real time PCR and HRM specify the analysis settings See the real time PCR instrument manual for details Note Real time cyclers not listed in this handbook often do not have the ability to perform HRM analysis Type it HRM PCR Handbook 07 2009 Q fr 5 z ro fe 5 7 jo siskjpuy Table 2 Optimized cycling protocol for HRM analysis on the Rotor Gene Q and Rotor Gene 6000 N 5 6 Step Time Temp Additional comments 2 Initial PCR 5 min 95 C HotStarTaq Plus DNA Polymerase is E activation step activated by this heating step lt 5 3 step cycling Important Optimal performance is only Lo assured using these cycling conditions Denaturation 10s 95 C Annealing 30 s 55 C Extension 10s 72 C Activate fluorescence data acquisition on the green channel Suitable for PCR products up to 3
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