qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
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Understanding Melt Curves for Improved SYBR® Green Assay Analysis and Troubleshooting
1. Understanding Melt Curves for Improved SYBR®
Assay Analysis and Troubleshooting
April 2, 2015
Dr Nick Downey, Applications Scientist
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INTEGRATED DNA TECHNOLOGIES
Outline
• Review of intercalating dye–based qPCR
• Theory of melt curves
• How melt curves can help diagnose problems
• Use of UmeltSM software to help with data interpretation
• Troubleshooting SYBR® dye–based experiments
• Steps to successful qPCR design
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INTEGRATED DNA TECHNOLOGIES
Intercalating Dye Assays vs. 5′ Nuclease Assays
Intercalating Dye Assays
• Inexpensive
• Non-specific PCR products and primer dimers will generate fluorescent signal
• Requires melting point curve determination
• Cannot multiplex
• Cannot be used for single-tube genotyping of 2 alleles
5′ Nuclease Assays
• 3rd sequence in assay (the probe) adds specificity
• Specific amplification for rare transcript or pathogen detection
• Does not require post-run analysis such as melt curves
• Can multiplex
• Can be used for single-tube genotyping of 2 alleles
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INTEGRATED DNA TECHNOLOGIES
SYBR® Green Dye
• Asymmetrical cyanine dye
• Intercalating dyes fluoresce only when bound
to DNA
• Most only bind efficiently to double-stranded DNA
• Similar cyanine dyes
• SYBR ® Green II
• SYBR Gold
• PicoGreen®
• DNA–dye complex:
• Absorbs blue light (λmax = 497 nm)
• Emits green light (λmax = 520 nm)
• Developed to quantify template (RNA and
DNA)
• Preferentially binds to double-stranded DNA
• Lower performance with single-stranded DNA
and RNA
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INTEGRATED DNA TECHNOLOGIES
Why Run Melt/Disassociation Curves When Using
Intercalating Dyes
SYBR® Green dye will detect any double-stranded DNA, including:
• primer dimers
• contaminating DNA
• PCR product due to mis-annealed primers
By viewing a dissociation/melt curve, you ensure that the desired
amplicon was detected
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INTEGRATED DNA TECHNOLOGIES
How Does a Melt Curve Help Data Analysis?
SYBR® Green assays detect any DNA; hence, the melt curve can indicate potential
issues, such as:
• gDNA contamination in an RNA sample
• Primer-dimers affecting the assay
• Splice variants (if there is extra sequence between primers)
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INTEGRATED DNA TECHNOLOGIES
Problem: Small Amount of gDNA in cDNA Sample
Assay targeting TCAF1 (TRPM8 channel-associated
factor 1) produces a single peak
No RT control also produces a single peak
Sample
Ladder
–RT
NTC
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INTEGRATED DNA TECHNOLOGIES
Problem: Small Amount of gDNA in cDNA Sample
Assay targeting TCAF1 (TRPM8 channel-associated
factor 1) produces a single peak
No RT control is necessary for diagnosing genomic DNA contamination.
No RT control also produces a single peak
Sample
Ladder
–RT
NTC
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INTEGRATED DNA TECHNOLOGIES
Problem: Large Amount of Contaminating gDNA
Sample Results No Reverse
Transcription
Assay across intron of BAIAP3 (BAI1-associated protein 3)
–RT
Sample
Ladder
NTC
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INTEGRATED DNA TECHNOLOGIES
Problem: Large Amount of Contaminating gDNA
Sample Results No Reverse
Transcription
Gel analysis confirms genomic DNA amplification
Assay across intron of BAIAP3 (BAI1-associated protein 3)
–RT
Sample
Ladder
NTC
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Melt Curves Show Removal of Off-Target Amplicons
RNA retreated with DNase
(BAIAP3 amplification)
Original RNA sample
(BAIAP3 amplification)
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INTEGRATED DNA TECHNOLOGIES
Not All Primer Dimers are a Problem for an Assay
Assay designed against PPIA, within a single exon
NTC shows multiple peaks, raising concern
about primer-dimers
CE analysis
indicates no
problem from
primer dimers
–RT
Sample
Ladder
NTC
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INTEGRATED DNA TECHNOLOGIES
Problem: Assay Designed Across a Small Intron
Low DNase High DNase gDNA
High DNase treatment does not resolve the issue
Possible solution: Probe-based assay across exon junction
LowDNase
HighDNase
LowDNase–RT
HighDNase–RT
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INTEGRATED DNA TECHNOLOGIES
Wittwer Lab is Interested in Understanding Melt Curves
• Designed a series of amplicons spanning exons of cystic fibrosis
transmembrane receptor (CFTR)
• Tested each one for melt characteristics and gel mobility
• Developed a model for melting of amplicon DNA
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INTEGRATED DNA TECHNOLOGIES
Extra Peaks in Melt Curves Do Not Always Indicate a Problem
Amplicon from exon 17b of CFTR Amplicon from exon 7 of CFTR
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Agarose Gel Electrophoresis is Useful for Confirming Melt
Curve Data
100 bp
200 bp
A B
Replicates of the
amplification of
CFTR exon 17b
Replicates of the
amplification of
CFTR exon 7
Gel electrophoresis is the
best method for analyzing
PCR products, but is very
labor- and time-consuming.
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INTEGRATED DNA TECHNOLOGIES
Best Methods for Assessing SYBR® Green Melt Curves
• Gold standard: gel electrophoresis
• Alternative: predict if melt occurs with more than one phase
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INTEGRATED DNA TECHNOLOGIES
uMeltSM Software Helps to Predict Melting of a PCR Product
uMeltSM predicts melt behavior of PCR
products:
https://www.dna.utah.edu/umelt/um.php
Developed by Wittwer lab
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INTEGRATED DNA TECHNOLOGIES
uMeltSM Software Predicts Melting of CFTR Exon 7 Amplicon
Different prediction
models are available
You can further
manipulate conditions
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INTEGRATED DNA TECHNOLOGIES
Troubleshooting SYBR® Green qPCR Assays
Observation/Problem Possible Cause Solution
Extra peaks in melt curves
Primer dimers
a. Decrease primer concentration
b. Increase annealing temperature
c. Redesign primers
Contamination
1. Template contaminated with gDNA
2. (bacterial target amplification) DNA
polymerase in master mix contaminated
with bacterial DNA
1. a. Run “– RT” control
b. Treat RNA template with DNase I
or design primers to span exons
2. Try new master mix
AT-rich subdomains causing uneven melting
a. Assess amplicon using uMeltSM tool
b. Run a gel to verify single product
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INTEGRATED DNA TECHNOLOGIES
Troubleshooting SYBR® Green qPCR Assays
Observation/Problem Possible Cause Solution
Poor amplification
Reagent missing from assay Repeat experiment
Annealing temperature too low Increase annealing temperature
Detection temperature needs
adjustment
a. Set temperature of detection to be below
amplicon Tm, but above Tm of primer dimers
b. Set detection reading at the annealing step
Amplicon is too long
Amplicons longer than 500 bp are not
recommended. Adjust extension time, if
necessary
Enzyme is not activated
Follow enzyme activation time based on master
mix
Template concentration too low Use template concentration up to 500 ng
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Steps for Designing a Reliable Assay
1. Know your gene.
2. Determine how many transcripts are associated with that gene.
3. Identify exons that are common or specific between the transcripts.
• Obtain a RefSeq accession number
• Use NCBI databases to identify exon junctions, splice variants, SNP locations
4. Align related sequences.
• For splice-specific designs:
• Identify unique regions within which to design primers and probe
• Avoid sequence repeats
5. Perform BLAST searches of primer and probe sequences.
• Ensure no cross reactivity with other genes within the species
6. Ensure that primers are not designed over SNPs.
7. Run the amplicon through the uMeltSM software to predict number of peaks.
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Primer Design Criteria
Melting temperature (Tm)
• Primer Tm values should be similar ±2C
• Normally ~60–62C
Length
• Aim for 1830 bases
GC content
• Do not include runs of 4 or more Gs
• GC content range of 35–65% (ideal = 50%)
Sequence
• Avoid sequences that may create secondary structures, self dimers, and heterodimers (IDT OligoAnalyzer® Tool )
Amplicon Length
• Ideal amplicon size: 80–200 bp
Design
• If measuring gene expression, design primers to span exon junctions
Always perform a BLAST search of potential primer sequences and
redesign if primer sequence is not target specific.
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Conclusions
• Intercalating dye use in qPCR is inexpensive and flexible.
• Observing the DNA melt dynamics of the amplicon via dye binding can be a useful tool for
distinguishing good data from bad.
• Take care when interpreting melt data due to the potentially complicated nature of melting.
• Before doing qPCR, get to know your gene and optimize assay and primer design.
• uMeltSM software is a useful online tool that can help you predict unexpected melt dynamics.
Animated: I’ll describe this occurs at the end of the amplification. Temp slowly increases with continuous monitoring of fluorescence. As the temp reaches the functional Tm of the DNA molecule it will begin to melt. As it does the SYBR will stop binding and will stop fluorescing.
Animated: Demonstration of a real trace. Initial description of loss of fluorescence and subsequent derivation of line (-delta slope). Indicate that we utilize the peak as a diagnostic point
Although there are single peaks in these data, looking closely indicates a shift in the temperature for the peak. CE analysis confirms this is due to a larger amplicon from gDNA.
Although there are single peaks in these data, looking closely indicates a shift in the temperature for the peak. CE analysis confirms this is due to a larger amplicon from gDNA.
Animated: example of unexpected result. Two peaks. Control experiment indicates a problem, no RT is also showing amplicon. CE results show a larger fragment (gDNA) is amplified
Animated: example of unexpected result. Two peaks. Control experiment indicates a problem, no RT is also showing amplicon. CE results show a larger fragment (gDNA) is amplified
Animated: demonstration that amplification is delayed after RNA is treated with DNase
Animated: demonstration that amplification is delayed after RNA is treated with DNase
New melt curve demonstrates single peak indicating sample is no lacking contaminating gDNA
Animated: assay shows single peak but NTC indicates multiple peaks. Sometimes it is inferred that this indicates off target issues. But CE shows that the peaks are due to primer dimers