Overview of Primer Probes in Realtime PCR
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.
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.
References:
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
Got something to say about this post? Leave a comment…your comments are valuable for improving the posts.