FAQ

This FAQ list is for MFEprimer-2.0. Please feel free to contact Wubin Qu (quwubin@gmail.com) for any questions or comments about MFEprimer-2.0.

What is MFEprimer?

MFEprimer is a program for checking the specificity of PCR primers based on multiple-factors, including sequence similarity, stability at the 3' end of the primer, melting temperature, GC content, and number of binding sites. It helps users to select more suitable primers before running either standard or multiplex PCR reactions. For more information please download and read this paper.

What is MFEprimer-2.0?

MFEprimer-2.0 is an unusual and remarkable updated version of MFEprimer. New features including:

1. High running speed, you can feel that. That's because we developed a new fast algorithm to replace the NCBI BLAST program for primer binding sites searching. This algorithm improves the running speed significantly.
2. Redesigned homepage and the result page. We used JavaScript and CSS technologies to redesign the homepage and try to improve the user experience.
3. Providing histograms of size, Delta G and Tm when number of predicted amplicons > 10.
4. Supporting degenerate primers (detect automatically).
5. Checking primer dimers and hairpins with ntthal program from Primer3 (http://primer3.sourceforge.net/).
6. Multiple database selection is allowed. This is useful for cross-species PCR primer analysis.
7. Added post-analysis functions, including: "ExportAmplicon" for exporting the amplicons in fasta-format; "MultiAlign" for multiple aligning the amplicons to check their similarity; "Cluster & MultiAlign" for amplicons clustering and multiple sequences alignment. These functions are useful when drawing a conclusion of the specificity of a PCR primer pair.
8. Besides the normal human-friendly output, the command-line version can output machine-friendly tab delimited formats. In addition, a Python parser (https://github.com/quwubin/MFEprimer/blob/master/chilli/MFEprimerParser.py) for normal output is also available for developers who want integrate MFEprimer into their own program, like a workflow.

Why should we need to check the specificity of the PCR primers?

Designing specific PCR primer is crucial for a successful PCR reaction. The non-successful results of a typical PCR reaction include non-specific products, smear bands or no any bands at all. The alternative products are mostly caused by non-specific PCR primers that amplify additional regions from the DNA template or the background DNA. So, it is essential to check the specificity of primers before PCR experiments. For additional information please visit Wiki.

How to choose the database?

Assuming that you want to amplify a human gene to detect whether it is expressed in certain condition, so your primers will be used to amplify the gene from the pool of all expressed human genes (here we call the background DNA/mRNA/cDNA). To avoid the nonspecific amplicons, you need to make sure that your primers won't bind to other genes. Here, you can choose "Human RNA" database for specificity checking. If the DNA samples may contain the genomic DNA fragments, you can choose the "Human - RNA & Genomic" for specificity checking. Actually, MFEprimer-2.0 acts as a virtual PCR, you can "SEE" the possible amplicons before running the real PCR reaction. Therefore, you can discard the nonspecific primers and choose a better PCR primer pair, as a result, saving your money and time.

The database of interest not available?

1. If the database is public, you can contact me (quwubin@gmail.com) and ask me to add it into the MFEprimer-2.0 server.
2. If the database is custom or private, you can 1) install the command-line version of MFEprimer; 2) build a local mirror of MFEprimer-2.0.

When to choose the multiple database?

When your background templates (gDNA/mRNA/cDNA) are complex and are from more than one species. This option should also be used if you want to know whether your PCR primers could be used to amplify both of the cDNA fragment and the relevant genomic DNA (gDNA) fragment of a same gene. In this case, the gDNA amplicon will be usually bigger than the relevant cDNA amplicon if there are introns in the gDNA region of the target gene.

When to use the batch mode?

• When you have to choose multiple database;
• When you check more than one PCR primer pairs, such as multiplex PCR primers. In this situation, primers should be in FASTA format (https://github.com/quwubin/MFEprimer/wiki/Fasta-Format).

How to treat the degenerate sequence?

MFEprimer-2.0 can automatically recognise the degenerate bases in your primer sequences and then convert them into normal sequences before running the program.

What is the purpose of Results filter settings?

Usually, MFEprimer-2.0 will output many predicted amplicons and these results will help you to evaluate the specificity primers. However, in some times, there are some predicted amplicons which have very low Tm values (explained below) and these amplicons would not be amplified in real PCR reaction due to the Tm difference between these amplicons and the target amplicon. Besides, too many amplicons bring troubles when evaluating the specificity of primers. In this situation, users can filter these low Tm amplicons and focus on the amplicons which will be probably amplified.

What is Tm?

Melting temperature.

What is Size?

The amplicon size predicted by MFEprimer-2.0.

What is PPC and what is PPC cutoff?

We defined primer pair coverage (PPC) to score the ability of the primer pair (a forward primer coupled with a reverse primer) binding to the DNA template using the following formula:

Where Fm and Rm are sequence overlaps of the forward primer and reverse primer with the DNA template, and Fl and Rl are the full lengths of the forward primer and reverse primer respectively. CVfr is the coefficient of variability of matched length of forward primer (Fm) and reverse primer (Rm). The maximum value of PPC is 100%, indicating a pair of primers with the same length and both of them binding to the template completely. See Fig. 1 and Fig. 2 below.

The PPC cutoff is an experiential value for removing the very unlikely happened amplicons in PCR reaction. However, this experiential value may not be proper according to different PCR conditions.

Fig. 1 PPC = 100.0%, one of the predicted amplicons of MFEprimer-2.0

Fig. 2 PPC = 21.6%, one of the predicted amplicons of MFEprimer-2.0

What's the purpose of Experimental settings?

The nearest-neighbor (NN) model was used to calculate melting temperature and Gibbs free energy. These settings are needed for the calculation. See Wiki for more information.

• Concentration of monovalent cations: The millimolar concentration of salt (usually KCl) in the PCR. MFEprimer-2.0 uses this argument to calculate primer melting temperatures. Default value is 50 mM.
• Concentration of divalent cations: The millimolar concentration of divalent salt cations (usually MgCl2) in the PCR. MFEprimer-2.0 converts concentration of divalent cations to concentration of monovalent cations using formula (7) suggested in the paper Ahsen et al., Clin Chem. 2001 Nov;47(11):1956-61. Default value is 1.5 mM.
• Concentration of dNTPs: The millimolar concentration of deoxyribonucleotide triphosphate. This argument is considered only if concentration of divalent cations is specified. Default value is 0.25 mM.
• Salt correction formula: MFEprimer-2.0 uses the salt correction formula (8) described in the paper SantaLucia 1998, DOI:10.1073/pnas.95.4.1460 for the melting temperature calculation.
• Annealing Oligo Concentration: The nanomolar concentration of annealing oligos in the PCR reaction. MFEprimer-2.0 uses this argument to calculate primer melting temperatures. The default (50nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research--0.5 microliters of 20 micromolar concentration for each primer oligo in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl2, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius. This parameter corresponds to 'c' in Rychlik, Spencer and Rhoads' equation (ii) (Nucleic Acids Research, vol 18, num 21) where a suitable value (for a lower initial concentration of template) is "empirically determined". The value of this parameter is less than the actual concentration of oligos in the reaction because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures.

What’s the MFEprimer-2.0 result and what’s the meaning of each section?

Note: This FAQ just only describes the output file (result) of MFEprimer-2.0 and what the meaning of each section is. For explaining the result, please click HowTo (https://github.com/quwubin/MFEprimer/wiki/HowTo) for detailed information. However, we still suggest users read this FAQ first to know much background information.

The MFEprimer-2.0 result contains 5 sections, including:

• Query: Fig. 3, the list of user input primer sequences with size, GC content and Tm values.
• Histogram of size, Tm and ΔG of xx potential amplicons: Fig. 3, an overview of the amplicons.
• Descriptions of xx potential amplicons: Fig. 3, the brief description of each predicted amplicon.
• Amplicon details: Fig. 4, the hybridization details information of each predicted amplicon.
• Parameters: Fig. 5, the parameters used in the evaluation process.
• Citation: Fig. 5, our first paper reports the MFEprimer program.

The first line of the result page begins with “MFEprimer-2.0 Report”, followed by several shortcut links for each section of the result by the red box in Fig. 3. Usually, the result page is probably very long and these links can guide users to the right section quickly.

The "Query" section lists the primer sequences input by the user one line per sequence named by “Seq1” and “Seq2” etc. For each primer sequence, we also calculated the size, GC content and Tm value as shown in Fig. 3.

The "Histogram of size, Tm and ΔG of xx potential amplicons" section providing histograms of size, Delta G and Tm when number of predicted amplicons > 10. So the users can see what the worst case scenario is for their primers and decide how to proceed (Thanks for the anonymous reviewer by providing this idea).

The "Descriptions of x potential amplicons" section lists all the predicted amplicons by MFEprimer-2.0 one line per amplicon. Several characteristics of each amplicon were also listed, including:

1. Fp x Rp: indicating that for this amplicon, which primer acts as a forward primer (Fp) and which one as a reverse primer (Rp).
2. Size: the size of this amplicon.
3. PPC: primer pair coverage (PPC), please go to FAQ PPC for detailed information.
4. Fp Tm: Tm value of the forward primer (Fp) in the binding pattern in this amplicon. Normally, the target amplicon has the highest Tm value, because the primer will bind perfectly with the target template. For those non-specific amplicons with some mismatches between the primer and its binding site, they usually have lower Tm values. If the Tm difference between the target amplicon and the non-specific amplicons is bigger than 10°C (an experiential cutoff value), the non-specific amplicon would not be amplified in the real condition which is the optimal condition for the target amplicon. This is the main theoretic basis of drawing a conclusion from the MFEprimer-2.0 result.
5. Rp Tm: Tm value of the reverse primer (Rp) in the binding pattern in this amplicon.
6. Fp ∆G: the Gibbs free energy between the forward primer and its binding site in this amplicon. The smaller the ∆G, the more stability the amplicon.
7. Rp ∆G: the Gibbs free energy between the reverse primer and its binding site in this amplicon.
8. Fp 3' ∆G: the Gibbs free energy between the last five bases of the 3' end of forward primer and its binding site in this amplicon.
9. Rp 3' ∆G: the Gibbs free energy between the last five bases of the 3’ end of reverse primer and its binding site in this amplicon.

Normally, these amplicons not only contain the target amplicon(s), but also the non-specific amplicon(s). So how to analyze the result and what’s the conclusion of the specificity of the primers? We maintain a special Wiki page named "HowTo" to help users under the MFEprimer-2.0 result. Please go to HowTo for further reading.

Fig. 3 Query and histogram sections of the MFEprimer-2.0 result

The "Amplicon details" section lists all the predicted amplicons by MFEprimer-2.0 just like the "Descriptions of x potential amplicons" section. The difference is that this section contains much more binding details of the amplicon as shown in Fig. 4. Some notes are added in red fonts in Fig. 4 to help users understanding the result. In this section, users can get an intuitive picture of the binding pattern between primers and the template sequence. The amplicon sequences in fasta format are useful for post-analysis, such as “MultiAlign?” for identifying the similarity of amplicons.

Fig. 4 Amplicon details section of MFEprimer result

The "Parameters" section contains parameters used in this evaluation. The meanings of each parameter were described in the previous FAQs.

The "Citation" section contains our first paper which reports the MFEprimer program. Although citing our paper in your manuscripts is not mandatory, it will help us to make the MFEprimer-2.0 better.

Fig. 5 Parameters and Citation sections of MFEprimer result

In MFEprimer-2.0, four post-analysis functions were added (Fig. 6). These functions were very useful to further analysis of the MFEprimer-2.0 results. For example, if a non-target amplicon has very close Tm value to the target amplicon, we might want to know the relationship of these two amplicons: splicing variants? gene duplication? or just another gene? In this situation, we can compare the sequences by the “MultiAlign” function. The “ExportAmplicon” function will let users export amplicons in fasta format for their own further analysis. In addition, the “Cluster & MultiAlign” function first clusters the amplicons by the similarity and then makes a multiple alignment for each cluster upon the request of users. The "Virtual Electrophoresis" function simulate the electrophoresis on a gel with 1% concentration, so users can judge whether the non-specific can affect the target amplicon in the final gel result.

These 5 sections contain all the information of each evaluation process, such as primers, results, parameters. Users can save the result page to an image file or PDF with third-party software for later use.

Fig. 6 Post-analysis functions

Questions not listed in this FAQ?

Please feel free to contact Wubin Qu (quwubin@gmail.com) or Chenggang Zhang (zhangcg@bmi.ac.cn).