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Tuesday, February 12, 2013

Primer and Probe Design for Real Time PCR - Quantitative Assays:

Primer and probe designing softwares are available online which can aid in easy designing of primer probe for the real time PCR quantitative assays. When designing only PCR primer pairs, other general primer design programs could be used such as:Primer Quest available free of charge at: and Primer Select by DNAstar.
Designing primers is one of the most important steps of a real-time PCR experiment. Several software packages are available to help with the design includingPrimer3, a free system from MIT . There are several considerations when designing real-time PCR primers. The product should be short, the two primers should have similar Tms (ideally within 0.5oC of each other but no more than 1 oC apart), and the primers should have low or no self complementarity (to avoid primer dimers). In addition, many people try to design primers that span introns or cross intron/exon boundaries. In this way, only cDNA from mRNA gene transcripts will be amplified, not genomic copies of the gene.

Overview of Primer Probes in Realtime PCR 

Primer Probe in real time PCR
                                                             Image Source

The guidelines for designing primers / probes for Quantitative assays:
· select the probe first and design the primers as close as possible to the probe without overlapping it. Amplicons of 50 to 150 bp are strongly recommended. If absolutely necessary product size could be increased up to 200-250bp at most
· keep primer/probe GC content within 30-80%
· avoid runs of identical nucleotides, this is especially true for guanine, where runs of four or more Gs should be avoided
When designing primers:
· Tm should be within 58°C to 60°C
· the last 5 bases at the 3' end should have no more than two G's or C's
· keep the annealing Ts of the primers as close as possible
· select primer pairs with minimal number of potential primer dimers and primer hairpins as possible
When designing probes:
· Tm should be within 68°C to 70°C
· no Gs on the 5' end
· select the strand that gives the probe more C than G bases

Before to proceeding with real time PCR, it is necessary to test the primers on a PCR reaction to ensure that the primers amplify the gene of interest at the right size and that the primers are specific (i.e. no other bands present on the gel except that of the expected size). In doing this, it is important to run your test PCR under conditions as close as possible to those for real time PCR . Also, the best results are achieved with magnesium concentration at 3mM and with optimal concentration of Taq.
Efficiency of the real time PCR quantitative assay

If perfect doubling occurs with each amplification cycle, the spacing of the fluorescence curves will be determined by the equation 2^n =dilution factor, where n is the number of cycles between curves at the fluorescence threshold (in other words, the difference between the Ct values of the curves).
for example, with a 10-fold serial dilution of DNA, 2^n = 10. Therefore, n = 3.32, and the Ct values should be separated by 3.32 cycles.

if the PCR assay has 100% efficiency one copy becomes two after one cycle, so how to calculate PCR Efficiency.

Amplification efficiency denoted by E can be calculated from the below equation:

E = 10^(-1/slope);

From the standard Curve chart (y = mx + b); 
click here to know how to generate a standard curve for a real time PCR quantitative assay

where m is the slope;
b is the intercept;

slope (m) = - 3.2746

Amplification efficiency E = 10^(-1/-3.2746) = 2.0

Amplification efficiency is also frequently presented as a percentage, that is, the percent of template that was amplified in each cycle.

To convert E into a percentage:

% Efficiency = (E – 1) x 100%

 % Efficiency = (2.0 – 1) x 100% = 100%.

An efficiency close to 100% is the best indicator of a robust, reproducible assay. Low reaction efficiencies < 90% may be caused by poor primer design or by suboptimal reaction conditions. Reaction efficiencies >100% may indicate pipetting error in your serial dilutions or co-amplification of nonspecific products, such as primer-dimers.

When using the method described above to determine amplification efficiency, the presence of inhibitor can also result in an apparent increase in efficiency. This is because samples with the highest concentration of template also have the highest level of inhibitors, which cause a delayed Ct, whereas samples with lower template concentrations have lower levels of inhibitors, so the Ct is minimally delayed. As a result, the absolute value of the slope decreases and the calculated efficiency appears to increase. If the  reaction efficiency is <90 % or >105%, one should modify the assay by redesigning your primers and probes.


PCR Application guide, Bio-Rad

Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR, Applied Biosystem

Mathematics in molecular biology and Biotechnology

Technical Resource, RRC Core Genomics Facility, University of Illinois at Chicago

Internet Sources