Understanding Melt Curves for Improved SYBR® Green Assay Analysis and Troubleshooting

Understanding Melt Curves for Improved SYBR®

Assay Analysis and Troubleshooting

April 2, 2015

Dr Nick Downey, Applications Scientist

2

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

3


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

4


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

5


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

6


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

7


Theory of Melt Curves

Temperature

Flu

ore

scen

ce

As the temperature is increased

the DNA starts to denature

8


The Initial Fluorescence Data is Manipulated to Produce a Quick Read Plot

9


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)

10


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

11


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

12


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

13


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

14


Solution: Treat RNA with More DNase

Original prep of RNA used for BAIAP3 (BAI1-associated protein 3) amplification

15


Solution: Treat RNA with More DNase

RNA for BAIAP3 amplification retreated with DNase

16


Melt Curves Show Removal of Off-Target Amplicons

RNA retreated with DNase

(BAIAP3 amplification)

Original RNA sample

(BAIAP3 amplification)

17


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

18


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

19


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

20


Extra Peaks in Melt Curves Do Not Always Indicate a Problem

Amplicon from exon 17b of CFTR Amplicon from exon 7 of CFTR

21


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.

22


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

23


A Model for Explaining the CFTR Exon 7 Double Peak

24


Best Methods for Assessing SYBR® Green Melt Curves

• Gold standard: gel electrophoresis

• Alternative: predict if melt occurs with more than one phase

25


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

https://www.dna.utah.edu/umelt/um.php

26


uMeltSM Software Predicts Melting of CFTR Exon 7 Amplicon

Different prediction

models are available

You can further

manipulate conditions

27


uMeltSM Dynamically Predicts Melt State

Slider controls temperature and animates dissociation along amplicon

28


uMeltSM Prediction Matches Melt Curve for CFTR Exon 13

100bp

200bp

29


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

30


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

31


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.

32


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.

33


Primer Assays from IDT for Human, Mouse, and Rat

34


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.

35


THANK YOU!We will email you the webinar recording and

slides next week.

Understanding Melt Curves for Improved SYBR® Green Assay Analysis and Troubleshooting

Science

Transcript of Understanding Melt Curves for Improved SYBR® Green Assay Analysis and Troubleshooting