QPCR analysis using qbase+

qbase+ introduction
2 Topics
Installation and Licence
Training Material
Experiment Design Exercises
7 Topics
Exercise 1: Simple Gene Expression Study
Exercise 2: A Large Study
Exercise 3: How to Fill Plates?
Exercise 4: A Growing Study
Exercise 5: A Diagnostic Copy Number Screen
Exercise 6: Fix Experiments with Bad or Missing Data
Exercise 7: Dilution Series for Calculating Amplification Efficiencies
Primer Design Exercises
5 Topics
Criteria for qPCR Primers
Fruit Fly Tap Gene: Designing qPCR Primers using PrimerBLAST
Fruit Fly Tap Gene: Analyzing primer characteristics using OligoAnalyzer
Human F9 Gene: Designing qPCR Primers using PrimerBLAST
Human F9 Gene: In Silico PCR in the UCSC Browser
Gene Expression Analysis
10 Topics
Loading Data
Adding Annotation to Data
Specifying the Aim of the Experiment
Checking the Quality of Technical Replicates and Controls
Considering Amplification Efficiencies
Normalization
Scaling
Visualizing the Results
Statistical Analysis
Run7
Analyzing Data from Different qPCR Experiments over Time
2 Topics
Creating a New Experiment
Analyzing the data
Selecting reference genes: exercises
8 Topics
About RefGenes
Starting the RefGenes Tool
The Genevestigator User Interface
Using the RefGenes Tool to Find Reference Genes
Finding Candidate Reference Genes in the Free Version of Genevestigator
Finding Candidate Reference Genes in the Commercial Version of Genevestigator
Exercise 1: Reference Genes for Mouse Liver
Exercise 2: Reference Genes for Human Heart
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Fruit Fly Tap Gene: Analyzing primer characteristics using OligoAnalyzer

OligoAnalyzer is a tool implemented by ID\&T (who sell primers) to check the characteristics of your primers. Take the first primer that is suggested by Primer-BLAST, the pair resulting in a product of 100bp.

What’s the Tm of the first primer ?
Copy the sequence of the first primer in the Sequence box, adjust the concentrations to these that are typically used in PCR (see slides) and click Analyze: As you can see the predicted melting temperature is 63.9 ºC, which is slightly different from the prediction made by BLAST. There are many different methods to predict Tm and each method will give a different result. Assumed concentrations of primers and ions have an enormous impact on the Tm prediction. So don’t worry about these differences: these are theoretical calculations anyway, the only way to determine Tm values is by doing actual PCR. As long as the difference in Tm between the two primers is not too large, everything is fine.
What’s the Tm of the second primer ?
Copy the sequence of the second primer in the Sequence box and click Analyze. The predicted melting temperature is also 63.9 ºC , the same Tm as the first primer.

Remember that the second primer had a large Self complementarity score according to PrimerBLAST.

Check the self-complementarity of the second primer in OligoAnalyzer ?
Click Self-Dimer: You see that the highest scoring alignment indeed has 6 matches, giving a score of 6 as predicted by PrimerBLAST.
Do you expect this self-complementarity will give problems in the PCR ?
No, the complementarity is concentrated at the center of the primer, not at the 3’ end. Since polymerases add bases at the 3’ end of the primer, the primer duplex cannot be extended so it will not give rise to aspecific products. ID&T recommends to avoid complementary stretches of more than 2 bp at the 3’ end. However, even if the primer dimer cannot be extended, it could interfere when its formation competes with the annealing of primer and target. This is only the case when the stability of the dimer is similar to the stability of a perfectly matched primer-target duplex. The stability of the perfectly matched duplex is shown as a Maximum Delta G at the top of results. So non-extendable dimer structures that are much shorter than the intended duplex, as we have here, are not going to disrupt the PCR reaction. It is advised to review all possible interactions between primers so both Self-Dimer (primers binding to themselves) and Hetero-Dimer (primers binding to each other) interactions between primers are examined.
Is it likely that the primers bind to each other ?
Click Hetero-Dimer:

This opens a text box to enter the second primer. Click Analyze. There is one structure (the fourth one) that looks problematic because there is a stretch of 3 matching nucleotides at the 3’end of one of the primers.

So you might consider taking a look at the second pair of primers that PrimerBLAST suggests. On the other hand, this structure is has relatively high free energy (delta G). The structure with the lowest total free energy, the target-primer duplex, is most important because it will dominate in solution. Structures with higher free energy are less stable and will be present in smaller amounts in the reaction mixture.

Take a look at the second primer pair that was suggested by PrimerBLAST.

Is it likely that these primers bind to each other ?
No these primers do not form duplex structures that could pose a problem during PCR.