understanding melt curves for improved sybr® green assay analysis and troubleshooting
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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
qPCR—Intercalating Dye vs. Probe-Based
Primers OnlyFor use with intercalating dyes such as
SYBR® Green
Primers and ProbeFor use in the 5’ nuclease assay
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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
Theory of Melt Curves
Temperature
Flu
ore
scen
ce
As the temperature is increased
the DNA starts to denature
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INTEGRATED DNA TECHNOLOGIES
The Initial Fluorescence Data is Manipulated to Produce a Quick Read Plot
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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 peakNo RT control also produces a single peak
Sam
ple
Lad
der
–R
TN
TC
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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
Sam
ple
Lad
der
–R
TN
TC
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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)
–R
T
Sam
ple
Lad
de
r
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)
–R
T
Sam
ple
Lad
de
r
NTC
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INTEGRATED DNA TECHNOLOGIES
Solution: Treat RNA with More DNase
Original prep of RNA used for BAIAP3 (BAI1-associated protein 3) amplification
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INTEGRATED DNA TECHNOLOGIES
Solution: Treat RNA with More DNase
RNA for BAIAP3 amplification retreated with DNase
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INTEGRATED DNA TECHNOLOGIES
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 AssayAssay designed against PPIA, within a single exon
NTC shows multiple peaks, raising concern
about primer-dimers
CE analysis
indicates no
problem from
primer dimers
–R
T
Sam
ple
Lad
der
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 issuePossible solution: Probe-based assay across exon junction
Low
DN
ase
Hig
h D
Nas
e
Low
DN
ase
–RT
Hig
h D
Nas
e –R
T
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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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INTEGRATED DNA TECHNOLOGIES
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
DNA Melting Is Not Always Biphasic
G-C-G-C-G-C-G-C-G-C-G-A-T-A-T-T-T-A-A-T-A-T-A
C-G-C-G-G-C-G-C-G-C-G-T-A-T-A-A-A-T-T-A-T-A-T
C-G-C-G-G-C-G-C-G-C-G-T-A-T-A-A-A-T-T-A-T-A-T
| | | | | | | | | | | | | | | | | | | | | | | G-C-G-C-G-C-G-C-G-C-G
C-G-C-G-G-C-G-C-G-C-G
| | | | | | | | | | |
Assumed eventPossible event
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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
uMeltSM Dynamically Predicts Melt State
Slider controls temperature and animates dissociation along amplicon
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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 concentrationb. Increase annealing temperaturec. Redesign primers
Contamination1. Template contaminated with gDNA
2. (bacterial target amplification) DNA polymerase in master mix contaminated with bacterial DNA
1. a. Run “– RT” controlb. Treat RNA template with DNase I
or design primers to span exons2. Try new master mix
AT-rich subdomains causing uneven meltinga. Assess amplicon using uMeltSM toolb. 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 longAmplicons longer than 500 bp are not recommended. Adjust extension time, if necessary
Enzyme is not activatedFollow enzyme activation time based on master mix
Template concentration too low Use template concentration up to 500 ng
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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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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INTEGRATED DNA TECHNOLOGIES
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.
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