Optimizing and Analyzing Real-Time Assays on the SmartCycler II System Cepheid Technical Support Overview This technical note provides guidelines on transferring, optimizing, and evaluating real-time PCR assays to the SmartCycler II System. Real-time PCR is far more complex than traditional PCR and offers a variety of detection chemistry options, such as DNA intercalating dyes or fluorescently-labeled probes such as TaqMan, Molecular Beacons, or Scorpions. Real time PCR offers several advantages over traditional PCR methods: Real-time monitoring Fluorescence signal and therefore temperature profiles, growth curves and melt curves are monitored in real time as data is collected. Rapid & Quantifiable results - By using one system to perform simultaneous amplification and detection, the need for post-amplification processing steps is eliminated. Reproducibility Quantitative methods based on active growth cycles are more consistent than traditional endpoint analyses. Here we discuss tips and guidelines to optimize SmartCycler PCR protocols. More detailed information on experimental design is covered in SmartNote 6.1, Designing Real-Time Assays on the SmartCycler II System. Optimizing SmartCycler II Protocols A typical PCR cycling protocol includes an initial denaturation step followed by cycling between a denaturation and an annealing step (2-step PCR) or a denaturation, annealing and extension step (3-step PCR). A 2-step protocol can be used if the annealing temperature is above 60 C. If the annealing temperature is below 60 C, real-time results might be suboptimal as the polymerase may not be completely active. For primers with T m less than 60, a three-step protocol is recommended. Initial denaturation: An initial hold stage at 95 C from 15 to 120 seconds to denature the DNA and activate Taq DNA polymerase. Note: Check the manufacturer s recommendations for activation time, as variation may exist based on the characteristics of the DNA polymerase. Denaturation: The first step in a two- or three-step protocol at 94 96 C for 3 15 seconds to denature the DNA at the start of each amplification cycle. Annealing: The second step in a two- or three-step protocol generally between 60 70 C for approximately 6 60 seconds. The optimal annealing temperature should be determined empirically and will depend on the melting temperature (T m ) of the primers and probe(s). The theoretical T m can be calculated using software available for free on the world wide web (refer to www.smartcycler.com for a list of PCR-related web links). Shorter annealing times favor specificity and minimize formation of primer-dimers. However, some primers require longer annealing times to obtain efficient amplification. We recommend designing primers that have an approximate T m between 60 C - 70 C. If possible, it is favorable to design primers with a T m that is at the higher end of this range to increase specificity. Extension: The third step in a 3-step protocol at 72 C for approximately 5-60 seconds, depending on DNA polymerase efficiency. Long amplicons (>400 bp) or primers with annealing temperatures below 60 C might require a 3-step protocol for optimum polymerase performance. defining on-demand molecular diagnostics.
Repeat cycles: The total number of cycles will depend on the initial amount of DNA and PCR efficiency. Each of the 16 I-CORE (Intelligent Cooling/Heating Optical Reaction) modules of the SmartCycler II processing block is independently controlled so that a different cycling protocol can be programmed for each reaction site. This allows the user to optimize cycle time and temperature simultaneously. When optimizing cycling parameters, we recommend varying one cycling step at a time. All cycling parameters should be examined when optimizing a protocol, including the initial hold and number of cycles. 1. Initial Hold: Test temperatures between 94 and 95 C and times between 15 and 120 seconds in 10-second increments. The whole time depends on the choice of thermostable DNA polymerase. 2. Denaturation: Test temperatures between 94 and 95 C and times between 3 and 15 seconds in 4- to 5-second increments. 3. Annealing/Extension: Use the table below as a guideline for simultaneous optimization of temperature and time. Temperature ranges will vary depending on the T m of the primer set. Time ranges will vary depending on the product size and PCR efficiency. Note: The optical read step must be at least 6 seconds. Extension: If separate annealing and extension steps are necessary, determine the optimal time and temperature in a similar manner. The table below represents a 16-sample experiment designed to determine the optimal temperature and time for a combined annealing/extension step. The calculated T m of the primer pair is 68 C and the denaturation step is held constant at 95 C for 5 seconds. Table 1: Example Matrix to determine optimal annealing temperature and time Site Time (sec.) C 1 40 66 2 30 66 3 20 66 4 10 66 5 40 64 6 30 64 7 20 64 8 10 64 9 40 62 10 30 62 11 20 62 12 10 62 13 40 60 14 30 60 15 20 60 16 10 60 Master Mix: Commercially available kits such as SmartMix HM can be used on the SmartCycler II System. SmartMix HM has been optimized specifically for the high surface to volume ratio of the SmartTube and the rapid thermal cycling capabilities of the SmartCycler. SmartMix HM is provided as a Smartbead that contains all of the necessary reagents, including Taq polymerase, dntps, MgCl 2 and HEPES, ph 8.0 buffer, for performing two 25µl reactions. When using SmartMix with your primers and probes we recommend resuspending in Tris ph 9.0 to achieve a final concentration of 10 mm. MgCl 2 : SmartCycler II assays often require more MgCl 2 than traditional PCR. We recommend varying the MgCl 2 from 2 to 6mM to determine the optimal concentration for your application. Remember that most 10X PCR buffers contain 15 mm MgCl 2. Too much MgCl 2 can lead to non-specific priming, whereas too little will result in suboptimal polymerase activity. However, Cepheid data has shown MgCl 2 concentrations as high as 8mM to be optimal when performing multiplex reactions. Note: MgCl 2 solutions can form concentration gradients over time when frozen and must be completely thawed and vortexed before use. Additives: The use of additives may enhance PCR productivity when using the SmartCycler II System. The unique design of the I-CORE reaction tube permits rapid cycling because of the high surface-to-volume ratio. However, the combination of high surface-to-volume ratio and rapid thermal cycling may reduce the end-point productivity of an amplification reaction. To increase reproducibility and productivity during the amplification process, the use of the SmartCycler Additive Reagent may be beneficial. Note: The additive reagent is not necessary if you are using SmartMix HM beads. Note: All additives must be tested empirically with specific template/primer combinations. Components of the SmartCycler Additive Reagent: We have found that a specific formulation of BSA, Trehalose, and Tween-20 has enhanced the productivity and sensitivity of several assays evaluated on the SmartCycler II System. BSA (Bovine Serum Albumin), non-acetylated aqueous solution (Sigma B-8667) Trehalose, from Saccharomyces cerevisiae, lyophilized, formula weight 378.3 (Sigma T-9531)
Tween-20 (Polyoxethylene Sorbitan Monolaurate, Pierce 28320 [product name is Surfact-Amps-20]) 1 M Tris Buffer, ph 8.0 (BioWhitaker 16-015Y) Note: Reagents, vendors, and product numbers specifically described above were used in all the testing of the Additive Reagent and should be used when preparing the formulation. Prepare a 1M Stock Solution of Trehalose: To make 10 ml of a 1 M stock concentration of trehalose, add 3.78g lyophilized trehalose to a sterile, RNase/DNase free 15 ml polypropylene vial. Dissolve the trehalose in 10ml 10 mm Tris buffer, ph 8.0. Prepare a 5X Stock Solution of the Additive Reagent: To produce 10 ml: 1. To a sterile, RNase/DNase free 15 ml polypropylene vial, pipette in 1 ml of 10 mm Tris buffer (ph 8.0). 2. Add 500 µl of 20 mg/ml non-acetylated BSA to the vial. 3. Add 7.5 ml of 1 M trehalose to the vial. 4. Add 1 ml 10% Tween-20 to the vial. 5. Mix thoroughly by vortexing. Table 2: Additive reagent compounds Component Bovine Serum Albumin (BSA) Stock Concentration Stock Volume 5X Stock Concentration Concentration in Final Reaction Mature (1X) 20 mg/ml 5 ml 1 mg/ml 0.2 mg/ml Trehalose 1 M 10 ml 750 mm 150 mm Tween-20 10% 10 ml 1% 0.2% Additive Reagent Storage: For long-term storage of the 5X stock, we recommend storing in 100 µl aliquots at -20 C. For frequent use, store the 5X stock at 4 C to prevent repeated freeze/thaws. The additive reagent is stable at 4 C for one week. Including the Additive Reagent in the PCR Master Mix: Add the additive reagent to the PCR master mix such that the final concentration is 1X. Please call technical support if you have any questions about incorporating the SC Additive into your PCR application. Optimizing the Master Mix for Real-Time PCR Probes: Dual-labeled probes such as TaqMan, Molecular Beacons or Scorpions are most frequently used on the SmartCycler II System. Dual-labeled probes are labeled with a reporter dye such as FAM on the 5 end and a quencher such as BHQ -1 on the 3 end. For optimal results, it is recommended to always design the probe before the primers. Below are some general recommendations for working with dual-labeled probes. Note: Avoid placing a G residue on the 5 end of a dual-labeled probe because it can have a quenching effect. T m (melting temperature): Design probe sequence (i.e. TaqMan, Molecular Beacons or Scorpions) with a T m of 5-10 C above the T m of the primers. Length: Probe length should be between 15 and 30 base pairs. Avoid designing a probe that is less than 15 base pairs. Purification: It is essential that real time PCR fluorescent labeled probes are as pure as possible to minimize background signal and to ensure that optimal results can be obtained. In general, many oligo vendors automatically perform de-salting purification for all oligos. However, a higher level of purification is recommended for real time PCR and any dye that requires an ester link. For more information on oligo purification methods see SmartNote 6.1, Designing Real-Time Assays on the SmartCycler II System. Dyes: The SmartCycler II System is calibrated for FAM, Intercalating dye, Cy3 or TET, Texas Red, Cy5, Alexa Flour 532, and Alexa Flour 647. Only calibrated dyes should be used for multiplexing. Quenchers: Dark quenchers such as Black Hole Quenchers (BHQ ), DABCYL, Eclipse or QSY -7 are recommended because fluorescence is not emitted when they are excited. Probes quenched by dark quenchers tend to have low background fluorescence and allow the use of all four channels for target detection. A TAMRA quencher is not recommended on the SmartCycler II because we have observed that TAMRA emits a strong signal in channel 2 and can trigger a warning (3079: Warning Fluorescence Too High). Storage: Always resuspend and store probes according to the manufacturer s recommendations. It is generally recommended to resuspend lyophilized probes to a 100µM stock solution in Tris ph 8.0 or nuclease-free H 2 0. Then, prepare a working stock at a final concentration between 10 and 25µM in Tris, ph 8.0 in 25 50 µl aliquots. Store all probes at -20 C protected from light to reduce degradation of the probe. Note: Resuspend Cy dyes (Cy3 and Cy5) in a non-amine containing buffer.
Caution: Avoid repeated freeze/thaw cycles of probes to reduce degradation of the probe. Concentration: The optimal concentration of every new probe must be empirically determined. The optimal assay concentration will depend on the amount of background signal, amplification/ binding efficiency and primer concentration. In general, the probe concentration can range from 0.1 0.4 µm. To minimize background fluorescence, always use the least amount of probe necessary to obtain adequate results. For a more complete, and chemistry-specific, list of recommendations for designing and working with dual labeled probes, see SmartNote 6.1, Designing Real-Time Assays on the SmartCycler II System. Primers: Ideally, primers should be designed after the probe and they should flank but not overlap the probe sequence. Primers should be designed to amplify as short an amplicon as possible for efficient amplification. In general, the primer length should be between 15 and 30 bases; however, longer primers increase specificity. It is recommended to design primers with a T m that is 5-10 C below the T m of the probe. Several factors should be considered when designing primers: GC content should be limited to 40 60% of the total sequence. Avoid complementary internal sequences to minimize formation of secondary structure. Avoid complementary sequences between the forward and reverse primer. Avoid a repeat of a single base, especially C s and G s at the 3 end. Design minimum product size for optimal PCR efficiency and minimum run time. If possible, design multiple forward and reverse primers that flank the probe region and test the various combinations of primers with the probe and pick the primer set that works best. example, if you are using too much primer, the primers may compete with the probe for binding and you will see gel results but no optical results. For more information about designing primers for realtime PCR experiments, see SmartNote 6.1, Designing Real-Time Assays on the SmartCycler II System. Intercalating Dyes: Intercalating dyes offer a low-cost alternative to visualize amplification in real time. An intercalating dye is a fluorescent molecule that increases in fluorescence when bound to double-stranded DNA. Intercalating dyes are often used for qualitative and semiquantitative PCR. Melt curve analysis is essential when you are using an intercalating dye for product discrimination and to determine the product purity based on the melting temperature (T m ). Although intercalating dyes can be used for semi-quantitative PCR, results will be more specific and accurate with a probe because fluorescence generated from a probe corresponds to a very specific product. Intercalating dye fluorescence represents all double-stranded DNA, including primer-dimers and other non-specific products. For more information about intercalating dyes, see SmartNote 6.4, Intercalating Dye Assays on the SmartCycler II System. Data Analysis When optimizing a real time PCR reaction, the growth curve should be monitored to determine when optimal conditions have been achieved. The growth curve should be sigmoidal (S-shaped) with three phases: baseline (background signal), log-linear (growth phase) and plateau. For each phase, the characteristics detailed below should be assessed. Figure 3: Sigmoidal Growth Curve After the primers and probe have been designed, the optimal annealing temperature, primer concentration, and probe concentration must be determined. The primer concentration can range from 0.1 µm to 1.0 µm; however, most applications will use between 0.2 and 0.5 µm. It is recommended to use the least amount of primer possible and to balance the primer and probe concentration. For
Baseline (background signal) The baseline phase of the growth curve represents initial cycles of amplification in which accumulation of specific signal has not exceeded the background signal. The fluorescent signal in this phase is from unbound probe, free dye, or auto fluorescing components in the reaction mixture. To monitor the background signal, turn the background subtraction OFF. This is located in the Analysis Settings in the View Results screen, you will need to click Update Analysis. The background signal should be as low as possible and should not exceed 500 fluorescent units at the beginning of the run. If the background signal is high at the beginning of the run, there is a greater chance that the signal will exceed the maximum calibration level and the overall results may be sub-optimal. Several factors can contribute to high background signal including: Free dye Auto fluorescing sample or reagent components Poor quenching of the reporter dye crosses a specified threshold is called the cycle threshold (C t ). The C t value can be used for qualitative or quantitative analysis. A qualitative analysis uses the defined threshold as a positive/negative measure. A quantitative assay uses the cycle threshold of defined standards of known concentration to generate a standard curve. Then, the cycle threshold values of unknown samples are used to interpolate the concentration(s) from the standard curve. The SmartCycler Software also allows determination of the cycle threshold by a mathematical analysis of the growth curve (rather than crossing a set threshold) by using the second derivative. The second derivative of the growth curve represents the rate of change in slope along the growth curve. The highest second derivative peak represents the greatest rate of change and signals the beginning of the log-linear phase. The cycle at which this peak occurs is designated as the cycle threshold. This analysis method can provide better run-to-run reproducibility than the primary signal analysis method. Probe concentration is too high Probe degradation Log-Linear: The log-linear phase of the growth curve represents the exponential amplification of target and will provide the most useful information about the reaction. The log-linear phase of the curve starts at the first significant increase in fluorescence above the baseline. The slope of the log-linear phase is a reflection of amplification efficiency. If there is no inflection point, the curve may not represent amplification of DNA, but rather signal drift. Drift is characterized by gradual increase or decrease in fluorescence without amplification of product. To check for drift, set the background subtraction to OFF in the Analysis Settings and check update analysis which will display the raw fluorescence data. If the raw fluorescence is high (>300) and the signal is linear, then the observed curve is drift, not a true growth curve. Plateau: The plateau phase of the growth curve occurs when critical components become rate-limiting and the accumulation of amplicon is no longer exponential. For this reason, endpoint measurements are often more variable and not optimal for quantitative measurements. Thresholds and Cycle Thresholds: Real time PCR offers the ability to utilize the log-linear phase of the growth curve for data analysis. This can provide a more accurate measurement than end point analysis. The cycle at which the growth curve
Real Time PCR Terminology Background Fluorescent signal derived from unbound probe, free dye, or sample auto fluorescence. Cycle Threshold If determined using the primary (fluorescent) signal, the cycle at which the signal crosses a user-defined threshold. If determined using the second derivative of the primary signal, the point of greatest rate change along the growth curve. Growth curve The curve generated by plotting the cycle number versus the measured fluorescent signal. Intercalating dye A non-sequence specific fluorescent dye that binds to double-stranded DNA. Primary signal The fluorescent signal. When plotted versus the cycle number, it produces the growth curve. T m The temperature at which 50% of the molecules are single-stranded and 50% are annealed to the complementary sequence. References Amersham Biosciences Product Specification Fluorolink Cy3-d-CTP PA 53021. http://www.amershambiosciences.com (18 July 2003). Annovis, 1999. Manual for the design and use of oligonucleotide specialty fluorescent probes. Perkin Elmer, 1998. TaqMan Universal PCR Master Mix Protocol, Rev A. http://molecular-beacons.org Behlke, M., Kelly, G., 2002. Dark Quenchers. Integrated DNA Technologies Technical Bulletins. Retrieved July 18, 2003, from http://www.idtdna.com/program/technical bulletin/ Dark_Quenchers. Behlke, M., Tao, W., 2002. Oligonucleotide Purification. Integrated DNA Technologies Technical Bulletins. Retrieved July 18, 2003, from http://www.idtdna.com/program/tech-nical bulletin/oligonucleotide_purification. Behlke, M., 2001. Resuspending Dry Oligos. Integrated DNA Technologies Technical Bulletins. Retrieved July 18, 2003, from http://www.idtdna.com/program/technical bulletin/ Resuspending_Lyophilized_Oligos. Bonnett, G., et al., 1999. Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc. Nat. Acad. Sci 96, pp. 6171 6176. Innis, M., Gelfand, D., Sninsky, J., 1999. PCR Applications: Protocols For Functional Genomics. Academic Press, San Diego, CA. www.cepheid.com CORPORATE HEADQUARTERS 904 Caribbean Drive Sunnyvale, CA 94089 USA toll free: 1.888.33743 phone: 1.408.541.4191 fax: 1.408.734.1346 EUROPEAN HEADQUARTERS Vira Solelh 81470 Maurens-Scopont France phone: 33.563.82.53.00 fax: 33.563.82.53.01 0192-02