Understanding Melt Curves for Improved SYBR®
Assay Analysis and Troubleshooting
Dr Nick Downey, Applications Scientist
April 2, 2015
Outline
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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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qPCR—Intercalating Dye vs. Probe-Based
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Primers Only
Primers and Probe
For use with intercalating dyes such as
SYBR® Green
For use in the 5’ nuclease assay
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Intercalating Dye Assays vs. 5′ Nuclease Assays
Intercalating Dye Assays
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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
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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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SYBR® Green Dye
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Asymmetrical cyanine dye
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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®
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DNA–dye complex:
•  Absorbs blue light (λmax = 497 nm)
•  Emits green light (λmax = 520 nm)
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Developed to quantify template (RNA and
DNA)
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Preferentially binds to double-stranded DNA
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Lower performance with single-stranded DNA
and RNA
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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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Theory of Melt Curves
Fluorescence
As the temperature is increased
the DNA starts to denature
Temperature
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The Initial Fluorescence Data is Manipulated to Produce a
Quick Read Plot
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How Does a Melt Curve Help Data Analysis?
SYBR® Green assays detect any DNA; hence, the melt curve can indicate
potential issues, such as:
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gDNA contamination in an RNA sample
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Primer-dimers affecting the assay
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Splice variants (if there is extra sequence between primers)
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Assay targeting TCAF1 (TRPM8 channel-associated
factor 1) produces a single peak
No RT control also produces a single peak
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– RT NTC Sample Ladder 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
No RT control is necessary for diagnosing genomic DNA contamination.
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– RT NTC Sample Ladder Problem: Small Amount of gDNA in cDNA Sample
Sample Results
No Reverse
Transcription
Assay across intron of BAIAP3 (BAI1-associated protein
3)
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NTC – RT Sample Ladder Problem: Large Amount of Contaminating gDNA
Sample Results
No Reverse
Transcription
Assay across intron of BAIAP3 (BAI1-associated protein
3)
Gel analysis confirms genomic DNA amplification
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NTC – RT Sample Ladder Problem: Large Amount of Contaminating gDNA
Solution: Treat RNA with More DNase
Original prep of RNA used for BAIAP3 (BAI1-associated protein 3) amplification
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Solution: Treat RNA with More DNase
RNA for BAIAP3 amplification retreated with DNase
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Melt Curves Show Removal of Off-Target Amplicons
Original RNA sample
(BAIAP3 amplification)
RNA retreated with
DNase (BAIAP3
amplification)
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Not All Primer Dimers are a Problem for an Assay
NTC shows multiple peaks, raising concern
about primer-dimers
NTC – RT Sample Ladder Assay designed against PPIA, within a single exon
CE analysis
indicates no
problem from
primer dimers
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High DNase treatment does not resolve the issue Possible solu?on: Probe-­‐based assay across exon junc?on 18
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High DNase –RT gDNA Low DNase –RT High DNase High DNase Low DNase Low DNase Problem: Assay Designed Across a Small Intron
Wittwer Lab is Interested in Understanding Melt Curves
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Designed a series of amplicons spanning exons of cystic fibrosis
transmembrane receptor (CFTR)
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Tested each one for melt characteristics and gel mobility
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Developed a model for melting of amplicon DNA
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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
A B Gel electrophoresis is the
best method for analyzing
PCR products, but is very
labor- and time-consuming.
200 bp
100 bp
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Replicates of the
amplification of
CFTR exon 17b
Replicates of the
amplification of
CFTR exon 7
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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
G-C-G-C-G-C-G-C-G-C-G
||||||||||||||||||||||
C-G-C-G-G-C-G-C-G-C-G-T-A-T-A-A-A-T-T-A-T-A-T
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C-G-C-G-G-C-G-C-G-C-G
Assumed event
A
-A-TT
A
-T-T-A
T
A
A-T
C-GG
C
C-GG
G-C
G-CC-G-C-G-G-C-G-C-G-C-G-T-A-T-A-A-A-T-T-A-T-A-T
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Possible event
A Model for Explaining the CFTR Exon 7 Double Peak
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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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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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uMeltSM Software Predicts Melting of CFTR Exon 7 Amplicon
Different prediction
models are
available
You can further
manipulate conditions
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uMeltSM Dynamically Predicts Melt State
Slider controls temperature and animates dissocia?on along amplicon 27
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uMeltSM Prediction Matches Melt Curve for CFTR Exon 13
200bp
100bp
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Troubleshooting SYBR® Green qPCR Assays
Observa(on/Problem Possible Cause Solu(on Primer dimers a.  Decrease primer concentra?on b.  Increase annealing temperature c.  Redesign primers Contamina?on 1.  Template contaminated with gDNA Extra peaks in melt curves 2.  (bacterial target amplifica?on) DNA polymerase in master mix contaminated with bacterial DNA AT-­‐rich subdomains causing uneven mel?ng 29
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1.  a. Run “– RT” control b. Treat RNA template with DNase I or design primers to span exons 2.  Try new master mix a.  Assess amplicon using uMeltSM tool b.  Run a gel to verify single product Troubleshooting SYBR® Green qPCR Assays
Observa(on/Problem Possible Cause Solu(on Reagent missing from assay Repeat experiment Annealing temperature too low Increase annealing temperature Detec?on temperature needs adjustment a.  Set temperature of detec?on to be below amplicon Tm, but above Tm of primer dimers b.  Set detec?on reading at the annealing step Amplicon is too long Amplicons longer than 500 bp are not recommended. Adjust extension ?me, if necessary Enzyme is not ac?vated Follow enzyme ac?va?on ?me based on master mix Template concentra?on too low Use template concentra?on up to 500 ng Poor amplifica?on 30
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Steps for Designing a Reliable Assay
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Know your gene.
Determine how many transcripts are associated with that gene.
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
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Align related sequences.
•  For splice-specific designs:
•  Identify unique regions within which to design primers and probe
•  Avoid sequence repeats
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Perform BLAST searches of primer and probe sequences.
•  Ensure no cross reactivity with other genes within the species
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Ensure that primers are not designed over SNPs.
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 ±2°C
•  Normally ~60–62°C
Length
•  Aim for 18-30 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
Always perform a BLAST search of potential primer sequences and
redesign if primer sequence is not target specific. •  If measuring gene expression, design primers to span exon junctions
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Primer Assays from IDT for Human, Mouse, and Rat
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Conclusions
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Intercalating dye use in qPCR is inexpensive and flexible.
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Observing the DNA melt dynamics of the amplicon via dye binding can be a useful tool for
distinguishing good data from bad.
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Take care when interpreting melt data due to the potentially complicated nature of melting.
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Before doing qPCR, get to know your gene and optimize assay and primer design.
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uMeltSM software is a useful online tool that can help you predict unexpected melt dynamics.
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THANK
YOU!
We will email you the webinar recording and slides next week. 35
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