Understanding Melt Curves for Improved SYBR Assay Analysis and Troubleshooting Dr Nick Downey, Applications Scientist April 2, 2015
Outline Review of intercalating dye based qpcr Theory of melt curves How melt curves can help diagnose problems Use of Umelt SM software to help with data interpretation Troubleshooting SYBR dye based experiments Steps to successful qpcr design 2
qpcr Intercalating Dye vs. Probe-Based 3 Primers Only For use with intercalating dyes such as SYBR Green Primers and Probe For use in the 5 nuclease assay
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 3 rd 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 4
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 5
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 6
Theory of Melt Curves As the temperature is increased the DNA starts to denature Fluorescence 7 Temperature
The Initial Fluorescence Data is Manipulated to Produce a Quick Read Plot 8
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) 9
Problem: Small Amount of gdna in cdna Sample Ladder Sample RT NTC Assay targeting TCAF1 (TRPM8 channel-associated factor 1) produces a single peak No RT control also produces a single peak 10
Problem: Small Amount of gdna in cdna Sample Ladder Sample RT NTC Assay targeting TCAF1 (TRPM8 channel-associated factor 1) produces a single peak No RT control also produces a single peak 11 No RT control is necessary for diagnosing genomic DNA contamination.
Problem: Large Amount of Contaminating gdna Sample Results No Reverse Transcription Ladder Sample RT NTC Assay across intron of BAIAP3 (BAI1-associated protein 3) 12
Problem: Large Amount of Contaminating gdna Sample Results No Reverse Transcription Ladder Sample RT NTC Assay across intron of BAIAP3 (BAI1-associated protein 3) Gel analysis confirms genomic DNA amplification 13
Solution: Treat RNA with More DNase Original prep of RNA used for BAIAP3 (BAI1-associated protein 3) amplification 14
Solution: Treat RNA with More DNase RNA for BAIAP3 amplification retreated with DNase 15
Melt Curves Show Removal of Off-Target Amplicons Original RNA sample (BAIAP3 amplification) RNA retreated with DNase (BAIAP3 amplification) 16
Not All Primer Dimers are a Problem for an Assay Assay designed against PPIA, within a single exon Ladder Sample RT NTC 17 NTC shows multiple peaks, raising concern about primer-dimers CE analysis indicates no problem from primer dimers
Problem: Assay Designed Across a Small Intron Low DNase High DNase gdna Low DNase High DNase Low DNase RT High DNase RT High DNase treatment does not resolve the issue Possible solu?on: Probe- based assay across exon junc?on 18
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 19
Extra Peaks in Melt Curves Do Not Always Indicate a Problem Amplicon from exon 17b of CFTR Amplicon from exon 7 of CFTR 20
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 21 Replicates of the amplification of CFTR exon 17b Replicates of the amplification of CFTR exon 7
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 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 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 22
A Model for Explaining the CFTR Exon 7 Double Peak 23
Best Methods for Assessing SYBR Green Melt Curves Gold standard: gel electrophoresis Alternative: predict if melt occurs with more than one phase 24
umelt SM Software Helps to Predict Melting of a PCR Product umelt SM predicts melt behavior of PCR products: https://www.dna.utah.edu/umelt/um.php Developed by Wittwer lab 25
umelt SM Software Predicts Melting of CFTR Exon 7 Amplicon Different prediction models are available You can further manipulate conditions 26
umelt SM Dynamically Predicts Melt State Slider controls temperature and animates dissocia?on along amplicon 27
umelt SM Prediction Matches Melt Curve for CFTR Exon 13 200bp 100bp 28
Troubleshooting SYBR Green qpcr Assays Observa(on/Problem Possible Cause Solu(on Extra peaks in melt curves Primer dimers Contamina?on 1. Template contaminated with gdna 2. (bacterial target amplifica?on) DNA polymerase in master mix contaminated with bacterial DNA AT- rich subdomains causing uneven mel?ng a. Decrease primer concentra?on b. Increase annealing temperature c. Redesign primers 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 umelt SM tool b. Run a gel to verify single product 29
Troubleshooting SYBR Green qpcr Assays Observa(on/Problem Possible Cause Solu(on Poor amplifica?on Reagent missing from assay Annealing temperature too low Detec?on temperature needs adjustment Amplicon is too long Enzyme is not ac?vated Template concentra?on too low Repeat experiment Increase annealing temperature a. Set temperature of detec?on to be below amplicon T m, but above T m of primer dimers b. Set detec?on reading at the annealing step Amplicons longer than 500 bp are not recommended. Adjust extension?me, if necessary Follow enzyme ac?va?on?me based on master mix Use template concentra?on up to 500 ng 30
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 umelt SM software to predict number of peaks. 31
32 Primer Design Criteria Melting temperature (T m ) Primer T m 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 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.
Primer Assays from IDT for Human, Mouse, and Rat 33
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. umelt SM software is a useful online tool that can help you predict unexpected melt dynamics. 34
THANK YOU! We will email you the webinar recording and slides next week. 35