Technical Note Dissociation Curve Analysis T08-003A General Introduction to Data Analysis The aim of this Technical Note is to explain the principle of dissociation curve analysis and to guide you through the experimental set up and analysis of data. It begins with a general introduction to data analysis. Results Editor The starting point for data analysis is the Results Editor. Before an analysis method has been set, the Results Editor home screen will display the plate layout and thermal cycling program. Each stage where readings have been made will have its own tab showing a graph of raw data. Figure 1: The Results Editor home screen. Each stage where readings have been made will have a separate results tab enabling analysis of the data for that particular stage. The Analysis Selection box displays the stage name as assigned in the program setup. Only those stages that have been assigned with reads are displayed since stages without reads have no data to analyse. Double-click on a stage name, or highlight a stage name then click on Edit to launch the Analysis Wizard Selection screen. Selecting the Analysis Method The Analysis Selection box on the Results Editor home screen allows the user to define the method of analysis to be applied to the readings gathered during the PCR run. Highlighting a stage name and pressing the Edit button will launch the Analysis Wizard Selection screen and allow an Analysis Method to be assigned for that stage. Figure 2: The Analysis Wizard Selection screen. Analysis Method: The drop-down menu lists analysis types appropriate to the selected stage. These will be dependent on the number of reads and the number of cycles programmed into the run. Dye name: The name of the dyes selected in the program setup will be displayed. Dye Usage: Assign a Dye Usage from the list in the drop-down menu. There will be one dye usage box for each read present in the stage. Cancel: Aborts the procedure and takes the user back to the Results Editor. Finish: Accepts all the default analysis settings for the analysis method chosen and closes the Wizard. Reset defaults: Returns all analysis parameter settings to the defaults. Back/Next: Allows the user to move between screens in the Analysis Wizards. The Analysis Wizards Once an analysis method has been defined, a series of default settings will automatically analyse the data. The defaults can be viewed in the Analysis Wizards and edited if required. The intuitive Analysis Wizards explain in detail the mathematics behind the analysis settings and will lead you through each stage of the analysis setup. With experience, analysis can be quickly performed using the Analysis Properties displayed in the Parameters (PAR) box feature found on each data graph. PrimeQHelp@bibby-scientific.com www.bibby-scientific.com +44(01785) 810433 1
Analysis Method Options The following analysis options are available in Quansoft: Baseline This simple analysis method allows for correction of differences in background fluorescence. It is also incorporated into many of the other analysis methods. Quantification Quantification analysis is used to determine the absolute or relative quantity of a target DNA template in a given test sample by measuring the cycle-to-cycle change in the fluorescent signal. The fluorescent signal increases proportionately to the amount of amplified DNA and quantification is performed either by comparison of the fluorescence of a PCR product of unknown concentration with that of several dilutions of an external standard, or by comparing the fluorescence of one product relative to another. To be able to make this comparison, the fluorophore is measured at a point in the amplification where the reaction efficiency can be considered optimal. This is generally around the cycle at which an increase in fluorescence is first detected. Dissociation curve Dissociation curve analysis can add to the information obtained from the PCR. Also known as melting curve analysis, it measures the temperature at which the DNA strands separate into single strands. This provides a measurement of the melting temperature or Tm, taken as the point at which 50% of the double stranded DNA (dsdna) molecules are dissociated. Using the easy-to-program ramp function, PrimeQ will perform a thermal ramping program that can be used to determine the Tm of the PCR product. This analysis provides the user with extra confidence in experiments using intercalating dye chemistry for identifying amplification of non-specific products or contamination. Plus-minus scoring This analysis exploits PrimeQ s fluorescence technology to determine with ease and accuracy the presence or absence of a PCR product in any given sample. Input data can either be kinetic (where readings are taken throughout the amplification stage) or end-point (readings taken at the end of the run). The software scores the samples as positive or negative according to user-defined thresholds. Allelic discrimination Users of PrimeQ have the option of this powerful technique capable of detecting single nucleotide differences (SNPs). It can be used to discriminate between genotypes, mutations and polymorphisms within or between samples simply by comparing the fluorescence signal obtained using allele-specific, dye-labeled probes. Multi-read This is a simple end point analysis method which will report the average fluorescence of a selected number of readings. It is useful for assays other than PCR, for example fluorescence-based DNA assays, where just the fluorescence of a sample needs to be measured; in this way PrimeQ can be used as a simple fluorescence plate reader or fluorimeter. PrimeQHelp@bibby-scientific.com www.bibby-scientific.com +44(01785) 810433 2
Analysis method: Dissociation Curve Dissociation curve analysis, also known as melting curve analysis, is used to determine the melting temperature (Tm) of a PCR product and uses intercalating dye chemistry. Intercalating dyes bind to the minor groove of doublestranded DNA (dsdna) producing up to a thousand-fold increase in fluorescence. Common examples of intercalating dyes include SYBR Green I, EvaGreen and BRYT Green. Figure 3: Principle of intercalating dye chemistry. The intercalating dye binds to dsdna. As the DNA is heated, the strands begin to dissociate and the dye is displaced causing the fluorescence to drop. The temperature at which 50% of the DNA is single stranded is the Tm. Since the Tm is influenced by GC content, length and sequence, it will be indicative of the identity of a PCR product. Dissociation curve analysis distinguishes PCR products on the basis of Tm. Since the Tm is characteristic of the GC content, length and sequence of a DNA product, it is a useful tool in product identification. This method is therefore commonly used to establish that the correct target DNA has been amplified and is most commonly used at the end of PCR assays to check for non-specific products and/or contamination. Figure 4: Identification of contamination or nonspecific products using Dissociation Curve analysis. Both A and B show amplification in the no template control (NTC) samples (blue lines). Following dissociation curve analysis, the melting peaks of the NTCs of A give a peak at the same Tm as the samples indicating the likelihood of contamination. In B, the peaks of the NTC products melt at a lower temperature suggesting that they are non-specific amplification products. Dissociation curve analysis also has applications in optimizing a PCR. If the Tm of a PCR product is known, the temperature at which the fluorescent data is collected can be programmed to be just below that of the specific PCR product, thus avoiding any detection of non-specific products. Also, peak area analysis, another feature of dissociation curve analysis, can give an estimation of the relative amount of each product since the size of the peak is generally proportional to the amount of amplification. PrimeQHelp@bibby-scientific.com www.bibby-scientific.com +44(01785) 810433 3
Setting up and running the experiment In order to perform dissociation analysis, the thermal cycling program needs to include a ramp stage. The Add Ramp button in the Program Editor allows setup of a dissociation curve stage whereby the temperature is raised in small increments between a defined range of temperatures; typically from the primer annealing temperature up to the system denaturation temperature (around 95 C). Fluorescence data is collected during each temperature increment after a defined hold time. Once the Tm of a particular PCR product is known, the temperature range of the ramp can be decreased to save time. Figure 5: Setting up a ramp stage to generate data for Dissociation curve analysis. If the Tm is unknown, start at a temperature just above the Tm of the primers and end at around 95 C. Once the Tm is known, the temperature range can be reduced and also the temperature increment can be reduced to give a more accurate value. During the run, the real-time collection of data can be monitored on the Run Screen. The plate layout shows the fluorescence curve on a per-well basis and the temperature profile plot indicates how far the run has progressed. When the run has completed, results can be viewed in the Results Editor with data from each stage of the run located under its own tab. Dissociation Curve Wizard setup Once the PCR has completed, open the results file and from the Results Editor home screen set up the Dissociation Curve analysis as described below. In the Analysis Selection box, highlight the stage name for analysis to be applied and click Edit. The Analysis Wizard will launch. Select Dissociation Curve from the drop-down menu in the Analysis Method selection box. Assign a dye usage for each of the reads (e.g. Reporter) and click Next. The Dissociation Analysis Wizard will launch. Figure 6: Analysis Wizard Selection. Select the Dissociation Curve analysis method and assign dye usages. For a Ramp stage, the only options will be Dissociation Curve or None. PrimeQHelp@bibby-scientific.com www.bibby-scientific.com +44(01785) 810433 4
Dissociation Curve Background Correction The first window of the wizard allows setting of background correction parameters. The background correction parameters are used to correct the data for the effects of temperature on fluorescence and for the background remaining after all the DNA has dissociated. The default is ON since this has to be enabled for dissociation curve analysis. Figure 7: Background Correction. The digital filter is an option to smooth the raw data before it is presented. It is carried out using a Savitsky-Golay curve smoothing algorithm, which fits a third order polynomial to a number of points either side of the data to be smoothed. Background cursors are placed in default positions. These can be updated when viewing the dissociation curve data. Peak Detection Clicking Next from the background correction screen leads to the peak detection options. The Dissociation Curve Wizard provides two options for peak detection: Auto peak detect: the software is instructed to automatically find a number of peaks mathematically. Manual peak detect: the user manually places cursors onto the graph of peak data to select the peaks. Figure 8: Auto Peak Detect. Number of peaks to find: Up to a maximum of four. Threshold filter: The user sets a threshold value to limit the size of the peaks detected (see below). Order by peak height or Tm: The results can be ordered according to either the peak height (largest first) or temperature (Tm). If there are replicates, each well will be treated individually. Figure 9: Manual Peak Detect. Number of peaks to find: Up to a maximum of four. Bin threshold: The user can specify a temperature range ( C) in which a peak will be considered valid. Threshold filter: The user sets a threshold value to limit the size of the peaks detected (see below). Once the data has been collected, the specific number of cursors will be present on the dissociation peak graph in the Results Editor. PrimeQHelp@bibby-scientific.com www.bibby-scientific.com +44(01785) 810433 5
Peak Area Once peak detection parameters have been set in the Dissociation Curve Wizard, clicking Next leads through to the Peak Area screen. Check the selection box for peak area calculation to be performed. The peak area option calculates the area under the selected peak(s) of interest and can provide an indication of the relative amounts of each product in a sample. The values will be displayed in the Results Table. Report Options and Summary The Report Options screen allows you to select data to appear in a printable report. The selection can be changed from the Report tab in the Results Editor if necessary. The Summary screen provides a quick checklist of the parameters set up in the Analysis Wizard. Click Back to change any settings or Cancel to abort the procedure. Click Finish to complete the set up. Viewing the analysed results Click on the results tab for the stage which has been set up for Dissociation Curve analysis. The analysed data will be displayed as a series of graphs similar to those shown in Figure 10. Individual graphs can be closed or enlarged for easier viewing. Clicking an individual well or a selection of wells in the plate layout will highlight just the selected well(s) on the graphs. Clicking the Show All Wells button will re-select all wells. Figure 10: The layout of data in the Results Editor screen for dissociation curve analysis. The Background Correction Graph Background correction is applied to a plot of fluorescence vs. temperature using two sets of user-defined cursors. The A cursors correct for the effect of temperature on the fluorescence signal which is not due to product melting. They should be positioned in the fluorescence region measured before any product has melted. The B cursors determine the remaining fluorescence background after the DNA has dissociated. They are placed in the flat fluorescence region that occurs after the melt. Click and drag the cursors to best suit the data, or set the position numerically by clicking on the PAR box next to the graph. Changing the position will automatically update the dissociation peak graph and results table. PrimeQHelp@bibby-scientific.com www.bibby-scientific.com +44(01785) 810433 6
Figure 11: Background correction cursors. The default values are: A1: Start temperature +10% ramp A2: Start temperature +30% ramp B1: Start temperature +90% ramp B2: Start temperature +95% ramp The Dissociation Peak Graph The dissociation peak graph is a plot of the negative first derivative of the dissociation curve and shows a characteristic peak for each product (the derivative is the negative of the rate of change in fluorescence as a fraction of temperature). By taking the derivative of the dissociation curve as opposed to using the raw data, identifying the Tm is made easier as a peak is produced. The Tm is the maximum point of the peak and is defined as the temperature at which 50% of the product has melted. Peak area analysis can also be performed and measures the area under a peak; this can provide an indication as to the relative amounts of each product. -df/dt 2,600 2,400 2,200 2,000 1,800 1,600 1,400 1,200 1,000 800 600 400 200 0-200 -400 72 74 76 78 80 82 84 86 Temperature 88 90 92 94 Figure 12: The dissociation peak graph. The peaks are derived from the background corrected data by calculating the negative first derivative of the dissociation curve. The peak corresponds to the Tm. Assignment of peaks If Auto peak detect is enabled, Quansoft will automatically find all peaks (up to a maximum of 4) in each sample which rise above the set threshold line. These will be presented in the Results table ordered according to either Tm or size (peak height). The Threshold filter appears as a red horizontal sliding cursor across the dissociation peaks graph at the default setting of 5%. This parameter limits the size of the peaks detected peaks smaller than the specified threshold % relative to the tallest peak are not recorded. The cursor can be moved manually to increase or decrease the threshold value for peaks detected. The new cursor position will be updated in the PAR box. PrimeQHelp@bibby-scientific.com www.bibby-scientific.com +44(01785) 810433 7
Figure 13: Auto Peak detect and Analysis Properties. The threshold line is shown in red. When Manual Peak Detect is selected, a cursor for each peak to find (up to 4) will be placed on the graph. To assign a set of peaks, drag each cursor to the centre of the peak(s). The Bin threshold is used to specify a temperature range ( C) either side of a cursor in which a peak will be considered valid. For example, if the cursor is placed at a temperature of 80 C and the Bin threshold set to 0.5 C, then all peaks with a Tm between 79.5 and 80.5 C will be considered valid and listed in the results table under that peak category. Figure 14: Manual Peak detect and Analysis Properties. Each cursor is used to label and classify a group of peaks. To help in classifying peaks, each peak cursor can be individually named by typing a name in the appropriate Peak Headings box. Click on the PAR button to view the Peak Headings box. Type in a name for the peak. The new name will then appear in the Results Table column heading and in the Report. This may facilitate identification of peaks corresponding to different PCR products. PrimeQHelp@bibby-scientific.com www.bibby-scientific.com +44(01785) 810433 8
Figure 15: The results table with Manual peak Detect. The table will give a separate column for each cursor, headed with the given name. All peaks which fall within the Bin threshold of the cursor will be listed in the table. Peak area Choose the Peak Area option by checking the box in the Analysis Properties. An additional column for each peak will be displayed in the Results table showing the area under the peak. This can give some indication of the relative amounts of PCR product, since the amount of amplification is related to the size of the peak. Figure 16: The results table with peak areas. PrimeQ Report If a printed report of the analysed data is required, open the Report tab of the Results Editor to view the Dissociation Curve report. To change any of the report contents, click on the Report Options icon to open up the Report Options box. Tabs will display the report options relevant for each stage. Change as appropriate and click Done to finish. The report can be printed or saved as a PDF file for future reference. Saving Saving will overwrite any previous analysis, therefore to preserve a particular analysis set up, click on File followed by Save As to save as a different file name. Trademarks ROX is a trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries. SYBR is a registered trademark of Life Technologies Corporation. EvaGreen is a registered trademark of BIOTIUM, INC BRYT Green is a registered trademark of Promega Corporation. PrimeQHelp@bibby-scientific.com www.bibby-scientific.com +44(01785) 810433 9