PCR vs Real-time PCR
PCR or Polymerase chain reaction is a revolutionized discovery in modern molecular biology, which was first developed by the chemist Kary Mullis in 1983. It allows a single sequence in a complex DNA to be amplified for analysis. The basic idea of the PCR is that two primers, which are complementary to the opposite strands of a DNA sequence, are oriented toward each other; the primers produce complementary strands, each containing the other primer. Hence, the result is a large quantity of a sequence corresponding to the DNA that lies between the two primers. DNA polymerase enzyme is used to extend the primers in PCR. DNA polymerase is a thermostable enzyme, and it has the ability to survive in high temperatures (94 to 95 °C) used for denaturation of template DNA.
The PCR involves three steps, namely, repeated rounds of denaturation, annealing of primers, and synthesis of DNA. A thermocycler machine is used to perform this reaction so that it can be programmed to change the temperatures quickly and accurately. Applications of the PCR are criminal investigations, DNA fingerprint, detection of pathogens, and analysis of DNA of early human species.
What is Conventional PCR?
There are three major stages of conventional PCR, namely; DNA amplification stage, separation of PCR, and detection of products. Separation of DNA segments are typically done by agarose gel electrophoresis. The products are then stained with etheiduim bromide. Finally, detection is achieved by visualization of bands onto gels under UV light. Therefore, the final results of conventional PCR are not expressed as numbers. Normally the conventional PCR is only able to detect a single parameter.
What is Real-time PCR?
Real-time PCR can detect the amplification of products, as the products are synthesized. With the development of technology, PCR has become a very popular technique, especially for the detection and identification of bacteria in foods. The real-time PCR uses a florescent dye system and thermocycler equipped with fluorescent- detection capability.
What is the difference between Conventional PCR and Real-time PCR?
• 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.