T08-003A Dissociation Curve Analysis - Techne · T08-003A: Dissociation Curve Analysis Setting up...

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Technical Note T08-003A

Dissociation Curve Analysis

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.

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.

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.

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.

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.

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T08-003A: Dissociation Curve Analysis

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.



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 double-

stranded DNA (dsDNA) producing up to a thousand-fold increase in fluorescence. Common examples of

intercalating dyes include SYBR® Green I, EvaGreen® and BRYT™ Green.

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.

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.

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.

Figure 4: Identification of contamination or non-

specific 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.



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.

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.

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.



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.

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 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.

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.



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.

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.

Figure 10: The layout of data in the Results Editor screen for

dissociation curve analysis.



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.

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.

Temperature949290888684828078767472

-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

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

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.



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.

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.

Figure 13: Auto Peak detect and Analysis Properties. The threshold line is shown in red.

Figure 14: Manual Peak detect and Analysis Properties. Each cursor is used to label and classify a group of peaks.



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.

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.

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.

Figure 16: The results table with

peak areas.

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