PyroMark_Q24_PCR_Han..
Contents
1. 10 bp 2 0 100 bp 110 bp 10 bp 10 bp 3 0 50 bp 100 bp 10 bp 10 bp PyroMark PCR Handbook 05 2009 19 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 qgiagen 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 PCR related errors Little or no product a HotStarTaq DNA Polymerase not activated b Pipetting error or missing reagent c High GC content d Primer concentration not optimal or primers degraded e Problems with starting template f Problems with bisulfite conversion reaction 20 Comments and suggestions Check whether PCR was started with an initial incubation step at 95 C for 15 min Repeat the PCR Check the concentrations and storage conditions of reagents including primers Use 12 5 ul PyroMark Master Mix per 25 ul reaction Using the same cycling conditions repeat the PCR using Q Solution Repeat the PCR with different primer concentrations from 0 1 0 5 uM of each primer in 0 1 uM steps However an excess of biotinylated primer might cause background in a Pyrosequencing assay2. Check the concentration storage conditions and quality of the starting template see Appendix A page 25 If necessary make new serial dilutions of template nucleic acid from stock solutions Repeat the PCR using the new dilutions Check the concentration storage conditions and quality of the bisulfite converted DNA Efficient bisulfite conversion and removal of PCR inhibitors is essential for optimal results Perform bisulfite treatment of nucleic acids from your sample using an appropriate bisulfite conversion We recommend the use of the EpiTect Bisulfite Kit for conversion PyroMark PCR Handbook 05 2009 Comments and suggestions g Primer design not We strongly recommend use of the PyroMark optimal Assay Design Software 2 0 for primer design h Incorrect denaturation Denaturation should be at 94 C for 30 to 60 s temperature or time Ensure that the initial 15 min 95 C incubation step was performed as described in step 6 of the PCR protocols i Incorrect annealing If possible perform annealing gradient from temperature or time 50 C to 65 C and check PCR products on an agarose gel Choose an annealing temperature that results in the highest specificity and yield If a gradient PCR is not possible decrease annealing temperature in 2 C steps Annealing time should be between 30 and 60 s i Mg concentration Perform PCR with different final concentrations of not optimal Mg from 1 5 5 0 mM in 0 5 mM steps u
3. Additional comments Initial PCR 15 min 95 C HotStarTaq DNA Polymerase is activation step activated by this heating step 3 step cycling Denaturation 30s 94 C Annealing 30s 60 C For genomic DNA 56 C For bisulfite converted DNA Extension 30s 72 C Number of cycles 45 Final extension 10 min 72 C Recommended annealing temperature when using PyroMark Assay Design Software 2 0 In all other cases 5 C below the calculated Tm of the primers is a suitable temperature to start with An annealing temperature that gives the highest specificity for the desired PCR product should be used 7 Place the PCR tubes in the thermal cycler and start the cycling program Note After amplification samples can be stored overnight at 2 8 C or at 20 C for longer storage 8 Use 5 20 ul of a 25 pl PCR for subsequent Pyrosequencing analysis PyroMark PCR Handbook 05 2009 13 Note In most cases 5 10 ul of the PCR product gives satisfactory Pyrosequencing results when using the PyroMark MD instrument 10 20 ul when using the PyroMark Q24 instrument and 20 ul when using the PyroMark ID instrument Adjust the amount of PCR product according to the instructions in the user manual of the specific instrument if required We recommend checking your PCR product prior to Pyrosequencing analysis e g by fast analysis on the QlAxcel or by agarose gel analysis PCR products can be directly loaded onto an agarose gel without prior add
4. and DNeasy systems for rapid purification of genomic DNA and viral nucleic acids and the RNeasy system for RNA preparation from a variety of sources For more information about QlAprep QlAamp DNeasy and RNeasy products contact one of our Technical Service Departments see back cover or visit www giagen com productfinder Quality of starting template when performing CpG assays Critical parameters for a successful PCR using bisulfite treated DNA template include complete bisulfite conversion and DNA fragments that are long enough for PCR The EpiTect Bisulfite Kit provides a fast and reliable procedure for efficient bisulfite conversion and prevents DNA fragmentation during the bisulfite conversion reaction by a unique DNA Protect Buffer For more information about EpiTect products contact one of our Technical Service Departments see back cover or visit www qiagen com Quantity of starting template The annealing efficiency of a primer to template is an important factor in PCR Owing to the thermodynamic nature of the reaction the primer template ratio strongly influences the specificity and efficiency of PCR and should be optimized empirically If too little template is used primers may not be able to find their complementary sequences Too much template may lead to an increase in mispriming events As an initial guide spectrophotometric and molar conversion values for different nucleic acid templates are listed in Tables 8 and 9 r
5. contains 3 mM MgCl and dNTPs Enzyme HotStarTaq DNA Polymerase is a modified form of a recombinant 94 kDa DNA polymerase Extension rate 2 4 kb min at 72 C Half life 10 min at 97 C 60 min at 94 C 5 3 exonuclease activity Yes Extra A addition Yes 3 5 exonuclease activity No Nuclease contamination No Protease contamination No RNase contamination No Self priming activity No Buffers and reagents Q Solution 5x concentrated MgCl solution 25 mM CoralLoad Concentrate 10x concentrated Contains gel loading reagent orange dye red dye SL_SL_LSSSSSS__EE_____E_E_E__E_ ___ _ _ _ _sss SSS Ss ee 6 PyroMark PCR Handbook 05 2009 Quality Control Enzyme Amplification efficiency assay PCR reproducibility assay Exonuclease activity assay Endonuclease activity assay RNase activity assay Protease activity assay Self priming activity assay Buffers and Reagents CoralLoad Concentrate 10x Q Solution 5x MgCl 25 mM RNase free water PyroMark PCR Handbook 05 2009 See quality control label inside kit lid for lot specific values The amplification efficiency is tested in parallel amplification reactions and is indicated under Amp PCR reproducibility and specificity are tested in parallel amplification reactions The reactions must yield a single specific product Linearized plasmid DNA is incubated with HotStarTaq DNA Polymerase in PCR Buf
6. an area separate from that used for DNA preparation or PCR product analysis M Use disposable tips containing hydrophobic filters to minimize cross contamination Procedure 1 Thaw PyroMark PCR Master Mix CoralLoad Concentrate primer solutions and Q Solution at room temperature or on ice It is important to mix the solutions before use in order to avoid localized concentrations of salt When using Q Solution additional MgCl is usually not required 2 Set up the reaction according to Table 5 It is not necessary to keep reaction vessels on ice since HotStarTaq DNA Polymerase is inactive at room temperature 16 PyroMark PCR Handbook 05 2009 Table 5 Reaction composition using PyroMark PCR Master Mix Component Volume reaction Final concentration Reaction mix PyroMark PCR Master Mix 12 5 ul Contains HotStarTaq DNA 2x Polymerase 1x PyroMark PCR Buffer and dNTPs CoralLoad Concentrate 2 5 pl 1x 10x Q Solution 5x 5 ul 1x Primer A Variable 0 2 uM Primer B Variable 0 2 uM RNase free water Variable Template DNA Template DNA added at Variable lt 500 ng reaction step 4 e 10 20 ng bisulfite converted DNA Total volume 25 ul Note If other reaction volumes are used reduce the amount of each component accordingly Contains 1 5 mM MgCl t Final primer concentration in the PCR reaction of 0 2 UM is normally optimal We recommend using 1 10 ng human genomic DNA If other DNA is used ad
7. cycles 22 Comments and suggestions We strongly recommend use of the PyroMark Assay Design Software 2 0 for primer design Repeat the PCR with different primer concentrations from 0 1 0 5 uM of each primer in 0 1 uM steps However an excess of biotinylated primer might cause background in a Pyrosequencing assay Check the concentration and storage conditions of the starting template see Appendix A page 25 Make serial dilutions of template nucleic acid from stock solutions Perform PCR using these serial dilutions We strongly recommend use of the PyroMark Assay Design Software 2 0 for primer design Repeat the PCR with different primer concentrations from 0 1 0 5 UM of each primer in 0 1 uM steps However an excess of biotinylated primer might cause background in a Pyrosequencing assay Perform PCR with different final concentrations of Mg from 1 5 5 0 mM in 0 5 mM steps using the 25 mM MgCl solution provided If the negative control PCR without template DNA shows a PCR product or a smear replace all reagents Use disposable pipet tips containing hydrophobic filters to minimize cross contamination Set up all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis The PyroMark PCR Master Mix is 2x concentrated Use 12 5 ul PyroMark Master Mix per 25 ul reaction Reduce the number of cycles in steps of 3 cycles PyroMark PCR Handbook 05 2009 Comments
8. length of the primers and by moving them back and forth Handling and storage of primers Determining primer concentration and quality Primer quality is crucial for successful PCR Problems encountered with PCR are frequently due to incorrect concentrations of primers being used If you observe problems in yield of amplification products check that primers were used at the correct concentration Storage buffer Lyophilized primers should be dissolved in a small volume of low salt buffer to give a concentrated stock solution e g 100 uM We recommend using TE buffer 10 mM Tris Cl 1 mM EDTA pH 8 0 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 28 PyroMark PCR Handbook 05 2009 Concentration Spectrophotometric conversion for primers 1 Aggo unit 20 30 ug ml To check primer concentration the molar extinction coefficient 260 can be used A260 E260 X molar concentration of primer or probe If the 54 value is not given on the data sheet
9. of the KRAS gene 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID For detection of mutations in codons 12 13 and 61 of the KRAS gene optimized for FFPE material 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID For detection of polymorphism in the APOE gene 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID For detection of variants in the MTHFR gene 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID PyroMark PCR Handbook 05 2009 Cat no 972022 972032 972052 972412 972402 972452 972422 972432 33 Product Contents Cat no PyroMark Q96 HFE For detection of variants in the HFE 972442 96 gene 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID PyroMark Q96 CpG For quantification of global methylation 973043 LINE 1 2 x 96 level in transposable elements only for use with PyroMark Q96 MD PyroMark Q24 KRAS PyroMark Q24 KRAS v2 0 PyroMark Q96 KRAS 96 and PyroMark Q96 KRAS v2 0 96 are intended for research use only Not for use in diagnostic procedures No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at ww
10. 13 and 61 of the KRAS gene optimized for FFPE material PyroMark Q96 Tests PCR primer and sequencing primer for quantification of methylation status and mutation detection PyroMark Q96 CpG pl 96 32 For quantification of methylation level in the p16 gene 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID PyroMark PCR Handbook 05 2009 Cat no 972812 972824 970012 970022 970032 970042 970412 970402 970452 972012 Product PyroMark Q96 CpG MLH1 96 PyroMark Q96 CpG MGMT 96 PyroMark Q96 CpG Prader Willi 96 PyroMark Q96 BRAF 96 PyroMark Q96 KRAS 96 PyroMark Q96 KRAS v2 0 96 PyroMark Q96 APOE 96 PyroMark Q96 MTHFR 26 Contents For quantification of methylation level in promoter region of the MLH1 gene 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID For quantification of methylation level in the MGMT gene 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID For quantification of methylation level in Prader Willi and Angelman Syndromes 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID For detection of mutations in exon 11 and mutation V6OOE in exon 15 as well as surrounding mutations in the BRAF gene 2 x 96 reactions on PyroMark Q96 MD and 1 x 96 reactions on PyroMark Q96 ID For detection of mutations in codons 12 13 and 61
11. May 2009 PyroMark PCR Handbook For PCR amplification of template DNA optimized for Pyrosequencing analysis QIAGEN Sample amp Assay Technologies 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 giagen com Contents Kit Contents Shipping and Storage Product Use Limitations Product Warranty and Satisfaction Guarantee Technical Assistance Safety Information Product Specifications Quality Control Introduction COON OH ua oH BR BB n Equipment and Reagents to Be Supplied by User Protocols B PCR Using PyroMark PCR Master Mix 11 B PCR Using PyroMark PCR Master Mix and Q Solution 15 Troubleshooting Guide 20 Appendix A Starting Template 25 Appendix B Assay and Primer Design Handling and Storage of Primers 26 Appendix C Control of Contamination 29 Ordering Information 31 PyroMark PCR Handbook 05 2009 3 Kit Contents PyroMark PCR Kit 200 800 Catalog no 978703 978705 Re
12. Most commercial bleach solutions are approximately 5 25 sodium hypochlorate FERE LL LLL LLLLLLLLLLLLLLLLLLLLLLILLLLLLLLLLLOILLLLLLLLLLLLLLLLLLLLCLLLLZECCUN 30 PyroMark PCR Handbook 05 2009 Ordering Information Product Contents Cat no EpiTect Bisulfite Kits for complete bisulfite conversion and cleanup of DNA for methylation analysis EpiTect Bisulfite Kit 48 EpiTect 96 Bisulfite Kit 2 48 EpiTect Bisulfite Spin Columns 59104 Reaction Mix DNA Protect Buffer Carrier RNA Buffers 2x EpiTect Bisulfite 96 well Plates 59110 Reaction Mix DNA Protect Buffer Carrier RNA Buffers EpiTect Control DNA for evaluation of PCR primers used for methylation analysis EpiTect Control DNA methylated 100 EpiTect Control DNA unmethylated 100 EpiTect Control DNA 1000 EpiTect PCR Control DNA Set 100 Methylated and bisulfite converted 59655 human control DNA for 100 control PCRs Unmethylated and bisulfite converted 59665 human control DNA for 100 control PCRs Unmethylated human control DNA for 59568 1000 control PCRs Human control DNA set containing 59695 both bisulfite converted methylated and unmethylated DNA and unconverted unmethylated DNA for 100 control PCRs PyroMark Gold Reagents for performing Pyrosequencing reactions PyroMark Gold Q24 Reagents 5 x 24 PyroMark Gold Q96 Reagents 5 x 96 PyroMark Gold Q96 Reagents 50 x 96 PyroMark PCR Handbook 05 2009 Nucle
13. PyroMark PCR Handbook 05 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 qiagen 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 you have any questions
14. action volume 25 pl 25 pl PyroMark PCR Master Mix 2x 3x0 85 12x0 85 ml ml CoralLoad Concentrate 10x 1x1 2ml Ax 1 2ml Q Solution 5x 1x2ml 4x2ml MgCl 25 mM 1x1 2ml Ax 1 2 ml RNase Free Water 1x1 9ml 4x 1 9 ml Handbook Includes HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl and dNTPs Shipping and Storage The PyroMark PCR Kit is shipped on dry ice but retains full activity at room temperature 15 25 C for 3 days The kit should be stored immediately upon receipt at 20 C in a constant temperature freezer When stored under these conditions and handled correctly this product can be kept at least until the expiration date see the inside of the kit lid without showing any reduction in performance Product Use Limitations The PyroMark PCR Kit is intended for molecular biology applications These products are neither intended for the diagnosis prevention or treatment of a disease nor have they 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 guidelines 4
15. and suggestions Pyrosequencing related errors Low or missing peaks in the Pyrosequencing run a Primer is insufficiently Ensure that PCR primers are of high quality use or not biotinylated HPLC purified biotinylated primers One of the primers must be biotin labeled to enable immobilization to streptavidin coated beads during preparation of a single stranded Pyrosequencing template b Sequencing primer Check that the sequencing primer binds to the does not match to the biotinylated strand biotinylated strand c Low quality Recommended PCR conditions will result in Pyrosequencing high quality Pyrosequencing templates in most template cases Optimize PCR if required see above However we recommend inspection of your PCR product on agarose gels Be sure to only use single banded templates with a sufficient yield for Pyrosequencing d Assay design not We strongly recommend use of the PyroMark optimal Assay Design Software 2 0 for designing PCR and sequencing primers to ensure correct primer design and orientation e Sample preparation Please refer to the user manual for the PyroMark and Pyrosequencing instrument which is used workflow related errors PyroMark PCR Handbook 05 2009 23 Comments and suggestions Poor or faulty Pyrosequencing run e Background of Check your PCR product on agarose gels to contaminating confirm that you only have one specific product sequence from PCR Additional sequence signals can be cau
16. creases PCR specificity in certain primer template systems Case C Q Solution has no effect on PCR performance Case D Q Solution causes reduced efficiency or failure of a previously successful amplification reaction In this case addition of Q Solution disturbs the previously optimal primer template annealing Therefore when using Q Solution for the first time for a particular primer template system always perform reactions in parallel with and without Q Solution M B l M D without QSolution 7194 bp 120 bp i wih Gi Solufion M morkers PyroMark PCR Handbook 05 2009 15 Important points before starting M When using Q Solution for the first time in a particular primer template system it is important to perform parallel amplification reactions with and without Q Solution E One primer must be biotinylated at its 5 end in order to prepare a single stranded PCR product for use in the subsequent Pyrosequencing procedure We recommend HPLC or an equivalent procedure to purify the biotinylated primer E For primer design we recommend use of PyroMark Assay Design Software 2 0 E The optimal PCR amplicon length for Pyrosequencing is between 80 and 200 bp although products up to 500 bp might work well for Pyrosequencing assays on genomic DNA M HotStarTaq DNA Polymerase requires an activation step of 15 min at 95 C see step 6 of this protocol E Setup all reaction mixtures in
17. e and optimization of agarose gel run time CoralLoad Concentrate can be added to the PCR without affecting amplification sensitivity or specificity PCR fragments amplified in the presence of CoralLoad Concentrate have been successfully tested for Pyrosequencing Q Solution The PyroMark PCR Master Mix is provided with Q Solution an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA This unique reagent will often enable or improve a suboptimal PCR caused by difficult templates that for example have a high degree of secondary structure or templates that are GC rich Unlike other commonly used PCR additives such as DMSO Q Solution is used at just one working concentration it is nontoxic and PCR purity is guaranteed PCR fragments amplified in the presence of Q Solution have been successfully tested for Pyrosequencing Specificity and sensitivity The PyroMark PCR Master Mix with its balanced potassium and sodium salts promotes specific primer template annealing and simultaneously reduces nonspecific annealing Maximum yields of specific products are obtained even when using extremely low template amounts r K XX s as PyroMark PCR Handbook 05 2009 9 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and p
18. e highest specificity for the desired PCR product should be used 7 Place the PCR tubes in the thermal cycler and start the cycling program Note After amplification samples can be stored overnight at 2 8 C or at 20 C for longer storage 8 Use 5 20 ul of a 25 pl PCR for subsequent Pyrosequencing analysis Note In most cases 5 10 ul of the PCR gives satisfactory Pyrosequencing results when using the PyroMark MD instrument 10 20 ul when using the PyroMark Q24 instrument and 20 ul when using the PyroMark ID instrument Adjust the amount of PCR product according to the instruments manual if required We recommend checking your PCR product prior to Pyrosequencing analysis e g by fast analysis on the QlAxcel or by agarose gel analysis PCR products can be directly loaded onto an agarose gel without prior 18 PyroMark PCR Handbook 05 2009 addition of a PCR loading buffer and gel tracking dyes when using CoralLoad Concentrate CoralLoad Concentrate contains a gel loading reagent and gel tracking dyes Refer to Table 7 below to identify the dyes according to migration distance and agarose gel percentage and type Note Due to the high viscosity of the solution apply the solution slowly into the wells of the agarose gel Table 7 Migration distances of gel tracking dyes TAE TBE agarose gel Red dye Orange dye 0 8 500 bp 270 bp 80 bp lt 10 bp 1 0 300 bp 220 bp 40 bp 10 bp 1 5 250 bp 120 bp 20 bp
19. espectively em s H J X SSS SSS ae Se PyroMark PCR Handbook 05 2009 25 Table 8 Spectrophotometric conversions for nucleic acid templates 1 Aygo unit Concentration ug ml Double stranded DNA 50 Single stranded DNA 33 Single stranded RNA 40 Absorbance at 260 nm 1 Table 9 Molar conversions for nucleic acid templates Nucleic acid Size pmol ug Molecules ug 1 kb DNA 1000bp 1 52 9 1 x 10 pUC19 DNA 2686 bp 0 57 3 4 x 10 Lambda DNA 48 502 bp 0 03 1 8x 107 Genomic DNA Escherichia coli 4 7x10 3 0x10 1 8 x 10 t Drosophila 1 4x10 1 1x10 6 6 x 10 melanogaster Mus musculus 27 X 10 cs ses 3 4 x 10 t mouse Homo sapiens 3 3x10 4 7x 10 2 8 x 10 human Base pairs in haploid genome t For single copy genes Appendix B Assay and Primer Design Handling and Storage of Primers Assay and primer design Primers should be designed using PyroMark Assay Design Software 2 0 The program automatically generates primer sets that include both PCR and sequencing primers Each primer set is given a quality score based on several parameters that are specific for Pyrosequencing analysis 26 PyroMark PCR Handbook 05 2009 PCR primer PCR primers should be 18 to 24 bases in length with annealing temperatures that are similar and typically in the range of 50 C to 68 C nearest neighbor method When using PyroMar
20. ever if a higher Mg concentration is required use of the 25 mM MgCl provided in the kit is recommended E Setup all reaction mixtures in an area separate from that used for DNA preparation or PCR product analysis E Use disposable tips containing hydrophobic filters to minimize cross contamination Procedure 1 Thaw the PyroMark PCR Master Mix CoralLoad Concentrate primer solutions and 25 mM MgCl if required at room temperature or on ice It is important to mix the solutions before use in order to avoid localized concentrations of salt 2 Set up the reaction according to Table 1 It is not necessary to keep reaction vessels on ice since HotStarTaq DNA Polymerase is inactive at room temperature Note The Mg concentration in the PyroMark Reaction Buffer will produce satisfactory results in most cases However in some cases reactions may be improved by increasing the final Mg concentration according to Table 2 PyroMark PCR Handbook 05 2009 11 Table 1 Reaction composition using PyroMark PCR Master Mix Component Volume reaction Final concentration Reaction mix PyroMark PCR Master Mix 12 5 ul Contains HotStarTaq DNA 2x Polymerase 1x PyroMark PCR Buffer and dNTPs CoralLoad Concentrate 2 5 pl 1x 10x 25 mM MgCl optional Variable see Table 2 See Table 2 Primer A Variable 0 2 uM Primer B Variable 0 2 uM RNase free water Variable Template DNA Template DNA added at Variable lt 500
21. fer Exonuclease activity per unit of enzyme is indicated under Exo Plasmid DNA is incubated with HotStarTaq DNA Polymerase in PCR Buffer Exonuclease activity per unit of enzyme is indicated under Endo RNA is incubated with HotStarTaq DNA Polymerase in PCR Buffer RNase activity per unit of enzyme is indicated under RNase HotStarTaq DNA Polymerase is incubated in storage buffer Protease activity per unit of enzyme is indicated under Protease Assays are performed under standard PCR conditions without primers HotStarTaq DNA Polymerase and human genomic DNA purified with the QlAamp DNA Blood Mini Kit The absence of PCR product is indicated by No under Self priming Conductivity and dye concentrations are tested Conductivity pH total aerobic microbiological count and performance in PCR are tested Conductivity total aerobic microbiological count and performance in PCR are tested Conductivity and RNase activities are tested Introduction The PyroMark PCR Kit is specifically optimized for Pyrosequencing analysis enabling highly specific and unbiased amplification of template DNA for various Pyrosequencing applications such as mutation detection SNP analysis methylation analysis and sequencing The convenient master mix format enables specific amplification of various starting materials using only one protocol Bisulfite treatment of genomic DNA converts nonmethylated cytosines to uracils leadi
22. ic PCR product PCR setup is quick and convenient as all reaction components can be combined at room temperature PyroMark PCR Master Mix The innovative master mix facilitates the amplification of specific PCR products from various starting materials such as genomic DNA from a variety of species as well as bisulfite converted DNA thereby generating PCR products that are highly suitable for Pyrosequencing During the annealing step of every PCR cycle the unique buffer composition ensures a high ratio of specific to nonspecific primer binding Owing to a uniquely balanced combination of KCI and NH SO the PCR buffer provides stringent primer annealing conditions over a wider range of annealing temperatures and Mg concentrations than conventional PCR buffers Optimization of PCR by varying the annealing 8 PyroMark PCR Handbook 05 2009 temperature or the Mg concentration is dramatically reduced and often not required CoralLoad Concentrate PyroMark PCR Kit is supplied with CoralLoad Concentrate which is strongly recommended to be used with the PyroMark PCR Master Mix for highly specific PCR and high yields of amplified DNA Furthermore PCR products amplified in the presence of CoralLoad Concentrate can directly be loaded onto an agarose gel without the need to add a gel loading buffer since it contains a gel loading reagent and two marker dyes an orange dye and a red dye that facilitate estimation of DNA migration distanc
23. ition of a PCR loading buffer and gel tracking dyes when using CoralLoad Concentrate CoralLoad Concentrate contains a gel loading reagent and gel tracking dyes Refer to Table 4 below to identify the dyes according to migration distance and agarose gel percentage and type Note Due to the high viscosity of the solution apply the solution slowly into the wells of the agarose gel Table 4 Migration distances of gel tracking dyes PyroMark PCR Handbook 05 2009 TAE TBE agarose gel Red dye Orange dye 0 8 500 bp 270 bp 80 bp lt 10 bp 1 0 300 bp 220 bp 40 bp 10 bp 1 5 250 bp 120 bp 20 bp 10 bp 2 0 100 bp 110 bp 10 bp 10 bp 3 0 50 bp 100 bp 10 bp 10 bp Protocol PCR Using PyroMark PCR Master Mix and Q Solution This protocol is designed for using Q Solution in PCR assays Q Solution changes the melting behavior of DNA and can be used for PCR systems that do not work well under standard conditions When using Q Solution the first time for a particular primer template pair always perform parallel reactions with and without Q Solution This recommendation should also be followed if another PCR additive such as DMSO was previously used for a particular primer template pair When using Q Solution depending on the individual PCR assay the following effects may be observed Case A Q Solution enables amplification of a reaction which previously failed Case B Q Solution in
24. just the amount according to the genome size see Appendix A page 26 3 Gently pipet the master mix up and down for thorough mixing and dispense appropriate volumes into PCR tubes 4 Add template DNA lt 500 ng reaction to the individual PCR tubes We recommend 10 ng human genomic DNA or 10 20 ng bisulfite converted DNA Note If the template is not human genomic DNA adjust the amount according to the genome size see Appendix A page 26 5 When using a thermal cycler with a heated lid do not use mineral oil Proceed directly to step 6 Otherwise overlay with approximately 100 pl mineral oil PyroMark PCR Handbook 05 2009 17 6 Program the thermal cycler according to the manvfacturer s instructions Note Each PCR program must start with an initial heat activation step at 95 C for 15 min Table 6 Optimized cycling protocol when using PyroMark PCR Master Mix and Q Solution Additional comments Initial PCR 15 min 95 C HotStarTaq DNA Polymerase is activation step activated by this heating step 3 step cycling Denaturation 30s 94 C Annealing 30s 60 C For genomic DNA 56 C For bisulfite converted DNA Extension 30s 72 C Number of cycles 45 Final extension 10 min 72 C Recommended annealing temperature when using PyroMark Assay Design Software 2 0 In all other cases 5 C below the calculated Tm of the primers is a suitable temperature to start with An annealing temperature that gives th
25. k Assay Design Software 2 0 for primer design an annealing temperature of 60 C for genomic DNA gives good results in most cases If you are not using PyroMark Assay Software 2 0 5 C below the calculated Tm of the primers is a suitable temperature to start with However the annealing temperature with highest specificity for the desired PCR product should be used One of the primers must be biotin labeled to enable immobilization of the PCR product to streptavidin coated beads during the preparation of single stranded Pyrosequencing template The orientation of the assay can either be forward or reverse The primer that needs to be biotinylated is indicated by the PyroMark Assay Design Software 2 0 The primers should not form strong hairpin loops or dimers with themselves or with the other primers The biotinylated primer should be carefully checked for hairpin loops and duplexes with the sequencing primer as excess biotinylated primer might cause background in Pyrosequencing assays If possible avoid placing primers over polymorphic positions The biotinylated primer should be purified by HPLC or an equivalent procedure since free biotin will compete with the biotinylated PCR product for binding on streptavidin coated beads Amplicon length The optimal amplicon length is between 80 and 200 bp although products up to 500 bp might work well Sequencing primer The sequencing primer should be 15 to 20 bases long and have an annealing
26. ng reaction step 4 e 10 20 ng bisulfite converted DNA Total volume 25 ul Note If other reaction volumes are used reduce the amount of each component accordingly Contains 1 5 mM MgCl t Final primer concentration in the PCR reaction of 0 2 UM is normally optimal We recommend using 1 10 ng human genomic DNA If other DNA is used adjust the amount according to the genome size see Appendix A page 26 Table 2 Final Mg concentrations Final Mg concentration 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 in reaction mM Required volume of 25 mM MgCl 0 05 1 15 2 25 3 3 5 per reaction wl 3 Gently pipet the master mix up and down for thorough mixing and dispense appropriate volumes into PCR tubes 12 PyroMark PCR Handbook 05 2009 4 Add template DNA lt 500 ng reaction to the individual PCR tubes We recommend 10 ng human genomic DNA or 10 20 ng bisulfite converted DNA Note If the template is not human genomic DNA adjust the amount according to the genome size see Appendix A page 26 5 When using a thermal cycler with a heated lid do not use mineral oil Proceed directly to step 6 Otherwise overlay with approximately 100 pl mineral oil 6 Program the thermal cycler according to the manvfacturer s instructions Note Each PCR program must start with an initial heat activation step at 95 C for 15 min Table 3 Optimized cycling protocol when using PyroMark PCR Master Mix
27. ng to DNA mainly consisting of three bases This less complex DNA is a challenging PCR template and the PCR products obtained are likely to have low yields Nonspecific products might be generated due to increased mispriming probability caused by the reduced complexity of DNA The unique master mix solution ensures specifically targeted primer binding and prevents mispriming due to a balanced combination of salts included in the PCR buffer leading to a specific PCR product An excess of biotinylated primer and artifacts generated during PCR can interfere with the Pyrosequencing procedure The PyroMark PCR Kit is optimized for use during Pyrosequencing analysis The specially formulated master mix prevents accumulation of an excess of biotinylated primer and minimizes generation of artifacts The high yields of specific PCR products obtained ensure reliable Pyrosequencing results HotStarTaq DNA Polymerase HotStarTaq DNA Polymerase is a modified form of the recombinant 94 kDa Taq DNA Polymerase from QIAGEN HotStarTaq DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures This prevents the formation of misprimed products and primer dimers at low temperatures HotStarTaq DNA Polymerase is activated by a 15 minute 95 C incubation step which can easily be incorporated into existing thermal cycling programs HotStarTaq DNA Polymerase provides high PCR specificity and often increases the yield of the specif
28. or experience any difficulties regarding the PyroMark 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 qiagen com 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 PyroMark PCR Handbook 05 2009 5 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 PyroMark PCR Master Mix 2x 2x concentrated Contains HotStarTaq DNA Polymerase and optimized PyroMark PCR Buffer
29. otides enzyme and substrate 978002 solutions intended for use with PyroMark Q24 For performing Pyrosequencing 972804 reactions on the PyroMark Q96 ID 5 x 96 and PyroMark Q96 MD 15 x 96 For performing Pyrosequencing 972807 reactions on the PyroMark Q96 MD and the PyroMark Q96 ID 31 Product PyroMark Gold Q96 SQA Reagents 1 x 96 PyroMark Gold Q96 CDT Reagents 6 x 96 Contents For performing Pyrosequencing reactions on the PyroMark Q96 ID For performing Pyrosequencing reactions on the PyroMark Q96 MD in combination with the capillary dispensing tips CDT PyroMark Q24 Tests PCR primer and sequencing primer for quantification of methylation status and mutation detection PyroMark Q24 CpG p16 4 x 24 PyroMark Q24 CpG MLHI 4 x 24 PyroMark Q24 CpG MGMT 4 x 24 PyroMark Q24 CpG LINE 1 4 x 24 PyroMark Q24 BRAF 4 x 24 PyroMark Q24 KRAS 4 x 24 PyroMark Q24 KRAS v2 0 4 x 24 For quantification of methylation level in the p16 gene For quantification of methylation level in the promoter region of the MLH Igene For quantification of methylation level in the MGMT gene For quantification of global methylation level in transposable elements For detection of mutations in exon 11 and mutation V6OOE in exon 15 as well as surrounding mutations in the BRAF gene For detection of mutations in codons 12 13 and 61 of the KRAS gene For detection of mutations in codons 12
30. rotective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier M Reaction tubes Pipets and pipet tips aerosol resistant Thermal cycler Mineral oil only if the thermal cycler does not have a heated lid Primers Primers should be purchased from an established oligonucleotide manufacturer Lyophilized primers should be dissolved in TE to provide a stock solution of 100 uM concentration should be checked by spectrophotometry Primer stock solutions should be stored in aliquots at 20 C see Appendix B page 28 SESE 10 PyroMark PCR Handbook 05 2009 Protocol PCR Using PyroMark PCR Master Mix Important points before starting E One primer must be biotinylated at its 5 end in order to prepare a single stranded PCR product for use in the subsequent Pyrosequencing procedure We recommend HPLC or an equivalent procedure to purify the biotinylated primer E For primer design we recommend use of PyroMark Assay Design Software 2 0 E The optimal PCR amplicon length for Pyrosequencing is between 80 and 200 bp although products up to 500 bp might work well for Pyrosequencing assays on genomic DNA M HotStarTaq DNA Polymerase requires an activation step of 15 min at 95 C see step 6 of this protocol M PyroMark PCR Master Mix provides a final concentration of 1 5 mM MgCl in the final reaction mix which will produce satisfactory results in most cases How
31. sed by miscellaneous events such as E Duplex formation of sequencing primer and or biotinylated primer E Hairpin formation of biotinylated primer and or template loop on Pyrosequencing template E Annealing between the sequencing primer and the biotinylated PCR primer M Nonspecific annealing of the sequencing primer to Pyrosequencing template Be sure to include following controls when you run an assays the first time M PCR without DNA M Sequencing primer on its own E Template without sequencing primer For more information see the user manual for the specific Pyrosequencing instrument We strongly recommend using PyroMark Assay Design Software 2 0 for designing PCR and sequencing primers suitable for Pyrosequencing ESSSSS___________E____ _ _ ___ _ __mnasss S SS Ss Se 24 PyroMark PCR Handbook 05 2009 Appendix A Starting Template Both the quality and quantity of nucleic acid starting template affect PCR in particular the sensitivity and efficiency of amplification Quality of starting template Since PCR consists of multiple rounds of enzymatic reactions it is more sensitive to impurities such as proteins phenol chloroform salts ethanol EDTA and other chemical solvents than single step enzyme catalyzed processes QIAGEN offers a complete range of nucleic acid preparation systems ensuring the highest quality templates for PCR These include the QlAprep system for rapid plasmid purification the QlIAamp
32. sing the 25 mM MgCl solution provided k Too much little The PyroMark PCR Master Mix is 2x master mix concentrated Use 12 5 ul PyroMark Master Mix per 25 ul reaction I Problems with the Check the power to the thermal cycler and that thermal cycler the thermal cycler has been correctly programmed m PCR overlaid with When performing PCR in a thermal cycler with a mineral oil when using heated lid do not overlay the PCR samples with a thermal cycler with a mineral oil if the heated lid is switched on as this heated lid may decrease the yield of PCR product Product is multi banded a PCR cycling conditions Using the same cycling conditions repeat the not optimal PCR using Q Solution Follow the protocol on page 15 b Annealing temperature f possible perform annealing gradient from ioo low 50 C to 65 C and check PCR products on an agarose gel Choose an annealing temperature that results in the highest specificity and yield If a gradient PCR is not possible increase annealing temperature in 2 C steps Annealing time should be between 30 and 60 s PyroMark PCR Handbook 05 2009 21 c Primer design not optimal d Primer concentration not optimal or primers degraded Product is smeared a Too much starting template b Primer design not optimal c Primer concentration not optimal or primers degraded d Mg concentration not optimal e Carryover contamination f Too much little master mix g Too many
33. supplied with the primers it can be calculated from the primer sequence using the following formula 260 0 89 x A x 15 480 C x 7340 G x 11 760 T x 8850 Example Concentration of diluted primer 1 uM 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 7340 6 x 11 760 6 x 8850 x 1 x 10 0 232 The measured A should be within 30 of the theoretical value If the measured A is very different to the theoretical value we recommend recalculating the concentration of the primers or having the primers resynthesized Storage of primers Primers should be stored in TE in small aliquots at 20 C Primers are stable under these conditions for at least one year Prepare small aliquots of working solutions containing 10 pmol ul to avoid repeated thawing and freezing Primer quality can be checked on a denaturing polyacrylamide gel a single band should be seen Appendix C Control of Contamination It is extremely important to include at least one negative control that lacks the template nucleic acid in every PCR setup to detect possible contamination General physical precautions E Separate the working areas for setting up the PCR master mix and DNA handling including the addition of starting template PCR product analysis or plasmid preparation Ideally use separate rooms E Use a separate set of pipets for the PCR mas
34. temperature in the range of 45 C to 55 C Ideally the sequencing primer should differ from the PCR primer by at least one additional specific base at the 3 end Specific considerations for CpG assays In CpG assays the optimal length of the primers is slightly increased to function on bisulfite treated DNA which has a high proportion of A and T PCR primers should usually be 22 30 bases and sequencing primers should be 18 23 bases in length When using PyroMark Assay Design Software 2 0 for primer design an annealing temperature of 56 C for bisulfite converted DNA gives good results in most cases The amplicons for CpG assays should ideally be shorter than 200 bp PyroMark PCR Handbook 05 2009 27 If sequences have densely packed CpG sites the design may require manual intervention However the penalties and scores given by the PyroMark Assay Design Software 2 0 are still very informative Manual operations for CoG assay design can include M Looking for the region of most interest and selecting it as the target M Placing the PCR primer without covering more than one CpG site M Ensuring the CpG site is in the 5 part of the primer if a primer needs to be located over a CpG site E Looking for warnings and checking for analysis parameters that can be improved Tm for primers mispriming duplexes between biotinylated primer and sequencing primer amplicon length etc E Trying to improve the final score by changing the
35. ter mix Use of pipet tips with hydrophobic filters is strongly recommended When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier PyroMark PCR Handbook 05 2009 29 E Prepare and freeze small aliquots of primer solutions Use of fresh distilled water is strongly recommended M Incase of contamination laboratory benches apparatus and pipets can be decontaminated by cleaning them with a 1 10 dilution of a commercial bleach solution t Afterwards the benches and pipets should be rinsed with distilled water General chemical precautions M PCR stock solutions can also be decontaminated using UV light This method is laborious however and its efficiency is difficult to control and cannot be guaranteed We recommend storing solutions in small aliquots and using fresh aliquots for each PCR E Another approach to preventing amplification of contaminating DNA is to treat individual reaction mixtures with DNase or restriction enzymes that cut between the binding sites of the amplification primers used before adding the template DNA sample When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier t
36. urt 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 giagen com 2009 QIAGEN all rights reserved www qiagen com Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 0086 21 3865 3865 Fax 0086 21 3865 3965 Technical 800 988 0325 800 988 0327 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 33430 420 Fax 02 33430 426 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 L
37. uxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 Spain Orders 91 630 7050 Fax 91 630 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 CLIII USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN Sample amp Assay Technologies
38. w giagen com or can be requested from QIAGEN Technical Services or your local distributor 34 PyroMark PCR Handbook 05 2009 Trademarks QIAGEN QlAamp GlAprep GlAxcel CoralLoad DNeasy EpiTect Pyrosequencing Q Solution RNeasy QIAGEN Group Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the PyroMark PCR Kit to the following terms 1 tnn Bp 9 The PyroMark PCR Kit may be used solely in accordance with the PyroMark 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 PyroMark PCR Handbook and additional protocols available at www qgiagen com 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 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 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 Co
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