Understanding Melt Curves for Improved SYBR® Green Assay Analysis and Troubleshooting
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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
- 2. 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. 3 INTEGRATED DNA TECHNOLOGIES qPCR—Intercalating Dye vs. Probe-Based Primers Only For use with intercalating dyes such as SYBR® Green Primers and Probe For use in the 5’ nuclease assay
- 4. 4 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
- 5. 5 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
- 6. 6 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
- 7. 7 INTEGRATED DNA TECHNOLOGIES Theory of Melt Curves Temperature Fluorescence As the temperature is increased the DNA starts to denature
- 8. 8 INTEGRATED DNA TECHNOLOGIES The Initial Fluorescence Data is Manipulated to Produce a Quick Read Plot
- 9. 9 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)
- 10. 10 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
- 11. 11 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
- 12. 12 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
- 13. 13 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
- 14. 14 INTEGRATED DNA TECHNOLOGIES Solution: Treat RNA with More DNase Original prep of RNA used for BAIAP3 (BAI1-associated protein 3) amplification
- 15. 15 INTEGRATED DNA TECHNOLOGIES Solution: Treat RNA with More DNase RNA for BAIAP3 amplification retreated with DNase
- 16. 16 INTEGRATED DNA TECHNOLOGIES Melt Curves Show Removal of Off-Target Amplicons RNA retreated with DNase (BAIAP3 amplification) Original RNA sample (BAIAP3 amplification)
- 17. 17 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
- 18. 18 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
- 19. 19 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
- 20. 20 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
- 21. 21 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.
- 22. 22 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 event Possible event
- 23. 23 INTEGRATED DNA TECHNOLOGIES A Model for Explaining the CFTR Exon 7 Double Peak
- 24. 24 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
- 25. 25 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
- 26. 26 INTEGRATED DNA TECHNOLOGIES uMeltSM Software Predicts Melting of CFTR Exon 7 Amplicon Different prediction models are available You can further manipulate conditions
- 27. 27 INTEGRATED DNA TECHNOLOGIES uMeltSM Dynamically Predicts Melt State Slider controls temperature and animates dissociation along amplicon
- 28. 28 INTEGRATED DNA TECHNOLOGIES uMeltSM Prediction Matches Melt Curve for CFTR Exon 13 100bp 200bp
- 29. 29 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
- 30. 30 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
- 31. 31 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.
- 32. 32 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.
- 33. 33 INTEGRATED DNA TECHNOLOGIES Primer Assays from IDT for Human, Mouse, and Rat
- 34. 34 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.
- 35. 35 INTEGRATED DNA TECHNOLOGIES THANK YOU! We will email you the webinar recording and slides next week.
Editor's Notes
- Review main points of the talk
- 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
- Approach differently, think about story.
- Heavily animated images