Real time PCR allows you to see exactly when and how the reactions become saturated. After so many cycles, the increase in DNA products is no longer linear. if you just examine bands on a gel, you do not know whether you still are in the linear range or not. The real-time machine gives you a readout after every cycle. Thus you can compare samples and be sure you are in the linear range of the assay.
Real-time PCR has several advantages over conventional PCR and vice-versa:
Real-time PCR can be used qualitatively or quantitatively, whereas conventional PCR is only qualitative (positive or negative). There are many occasions when it is important or helpful to know how approximately many copies of template DNA are present in the sample.
Real-time PCR is often more sensitive because this type of assay is designed to amplify short products which amplify more efficiently.
Real-time PCR can be more specific than conventional PCR. The additional specificity is provided by the specificity of the probe sequence.
Real-time PCR often has shorter cycle times than conventional assays because less time is required to extend across short amplicons.
Conventional assays offer advantages over real-time PCR, sometimes because they can easily be designed to be less specific when that is desirable, such as in consensus primer PCR to detect any member of a group of organisms.
Real time PCR (qPCR) is far more sensitive than end point PCR (conventional PCR). It will allow you to detect slight changes in expression between genes or samples and will allow you to analyze genes with very low expression.
qPCR could provide absolute quantification and relative quantification, while PCR is at most, semi-quantitative.
In short, if you want to detect presence/absence of a gene or if it is expressed or not, a conventional PCR will work just fine. If you want to quantify number of copies or the level of expression of a certain gene, qPCR is the way to go.
Real time PCR gives relative quantitative expression on the basis of detection of DNA by a flourescent dye ie. SYBR green in REAL TIME unlike conventional PCRs wherein the results can be interpreted at the end of the PCR reaction.
Real Time PCR is a more sensitive and accurate method for analyzing the quantitative & qualitative expression of particular genes at different set of experimental conditions or in different tissues or organisms. Conventional PCR gives qualitative expression of the presence of the gene.
In Real Time PCR, the quality of the run/amplification can be monitored during the run by analyzing the MELT CURVE and Ct values for controls and test samples whereas in conventional PCR the results can only be detected at the end of the reaction after the PCR products are loaded onto an Agarose gel with EtBr and visualized under UV light.
Real time PCR is less time consuming and labor intensive as compared to conventional PCRs
• Conventional PCR is more time consuming as it uses gel electrophoresis to analyze the amplified PCR products. In contrast, real-time PCR is less time consuming as it can detect amplifications during the early phases of the reaction.
• Real-time PCR collects data at the exponential growth phase of PCR while traditional PCR collects data at End-point of the reaction.
• The end point results of the conventional PCR may not be very precise, but the results of the real-time PCR are very precise.
• Real-time PCR is more sensitive than conventional PCR.
• Conventional PCR has very poor resolution while real-time PCR can detect very little changes due to the high resolution.
• End point detection of conventional PCR has short dynamic range while real-time PCR detection has wide dynamic range.
• Unlike conventional PCR, automated detection techniques are found in real-time PCR.
• Conventional PCR is highly sophisticated and labor intensive more than real-time PCR.
• Unlike real-time PCR, conventional PCR cannot discriminate between dead and live bacteria.
• Real-time PCR uses fluorescent dye system to detect the products while conventional PCR uses ethidium bromide and UV light to visualize bands in the agarose gel medium.