Understanding Melt Curves for Improved SYBR

Assay Analysis and Troubleshooting

Dr Nick Downey, Applications Scientist

April 2, 2015

Outline

Review of intercalating dyebased qPCR

Theory of melt curves

How melt curves can help diagnose problems

Use of UmeltSM software to help with data interpretation

Troubleshooting SYBR dyebased experiments

Steps to successful qPCR design

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qPCRIntercalating Dye vs. Probe-Based

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

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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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

DNAdye complex:
Absorbs blue light (max = 497 nm)
Emits green light (max = 520 nm)

Developed to quantify template (RNA

and DNA)

Preferentially binds to double-stranded

DNA

Lower performance with single-stranded

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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

Fluorescenc
e

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:

gDNA contamination in an RNA sample

Primer-dimers affecting the assay

Splice variants (if there is extra sequence between primers)

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Assay targeting TCAF1(TRPM8 channelassociated 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 channelassociated 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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NTC shows multiple peaks, raising

concern
about primer-dimers

NTC

RT

Sample

Assay designed against PPIA, within a single

exon

Ladder

Not All Primer Dimers are a Problem for an Assay

CE analysis
indicates no
problem from
primer
dimers

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High DNase treatment does not resolve the issue

Possible solution: Probe-based assay across exon junction

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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

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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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

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

-T-A
A
T
-AA
-T-T
T
T-A
G-C-G-C-G-C-G-C-G-C-G-A-

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
|||||
Assumed
event

CC-GG
- G- C

-T-A
C-G-C-G-G-C-G-C-G-C-G
-T-A
-AA-T
-T-A
-T-A
-T

-T-A
A
T
A
-T-T-A
T
A
G-A-T

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
dissociation
along

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uMeltSM Prediction Matches Melt Curve for CFTR Exon

13

200b
p
100b
p

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Troubleshooting SYBR Green qPCR Assays

Observation/Problem

Extra peaks in melt

curves

Possible Cause

Solution

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

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AT-rich subdomains causing uneven

melting
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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

Observation/Proble
m

Possible Cause

Solution

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

Poor amplification

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Steps for Designing a Reliable Assay

1.
2.
3.

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

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.
7.

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 2C
Normally ~6062C

Length
Aim for 1830 bases

GC content
Do not include runs of 4 or more Gs
GC content range of 3565% (ideal = 50%)

Sequence
Avoid sequences that may create secondary structures, self dimers, and heterodimers (IDT
OligoAnalyzer Tool )

Amplicon Length
Ideal amplicon size: 80200 bp

Design
perform design
a BLAST
search
of potential
primer sequences and
If measuring Always
gene expression,
primers
to span
exon junctions
redesign if primer sequence is not target specific.

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Primer Assays from IDT for Human, Mouse, and Rat

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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.

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THANK
We will email you the webinar
YOU!
recording and slides next week.
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