PCR & qPCR Plates

Polymerase Chain Reaction (PCR)

This is also called the standard PCR. It requires the DNA polymerase, nucleotides, primers,  DNA templates,  amplifier, thermocycler, amplified DNA, or deoxyribonucleic acid.  The standard PCR procedure consist of the following steps: 

• The double-stranded DNA is denatured with the heat
• The primer designing takes place which aligns with the single DNA strands
• Here, the primers are extended with DNA polymerase enzyme which results in two separate copies of the original DNA
• The denaturation, annealing, and renaturation process happens in series to compete for the synthesis of DNA hybridization and DNA replication
• A series of temperature and time is applied to the core steps of PCR. The cycle of amplification should be done to get the optimized DNA samples

These are also called steps of PCR by which the process executes. The PCR cycles are repeated almost 20-40 times and the amplified product is then analyzed using the thermal cyclers. The analysis is done to get the DNA detection in the sample of DNA.

The polymerase chain reaction is called the sensitive method.  So, it requires very small amounts of DNA to execute a single cycle of PCR. The single unit after the process of denaturation and annealing is differentiated into many small units to avoid any contaminations.

Reverse Transcriptase Polymerase Chain Reaction

In reverse transcriptase PCR, RNA acts as the template for the reaction.  In addition to all polymerase chain reaction steps,  a step of amplification of RNA is done to get the DNA aligned. The reverse transcriptase enzyme transcribes the RNA into complementary DNA (cDNA ). It is absent in the standard polymerase chain reaction.  The reverse transcriptase enzyme has the RNase H  which functions to degrade the RNA  position of the hybrid. After this, the next process is concluded by the DNA polymerase I enzyme.  The rest of the process of polymerase chain reaction is the same. However, the RNA strands are very sensitive, so they require more carefulness while handling it. Due to the involvement of the reverse transcriptase enzyme, it is called a reverse transcription-polymerase enzyme. When the pcr reaction is combined with quantitative pcr, then it is called real-time RT PCR. 

Quantitative Polymerase Chain Reaction

The quantitative polymerase chain reaction is also called real-time PCR.  in this pcr technique, it   gives out the idea of how much percentage of DNA is present in the sample.  In the above RT PCR technique, it is very cumbersome to detect the amount of DNA or RNA. But with the qPCR technique, it can be quantified.

PCR Vs QPCR

The standard PCR at one go can amplify the DNA up to 2000 nucleotides. While the QPCR   can amplify as well as quantify the DNA samples.  Here, the quantification is done to measure how many DNA samples are present in the sample.

The PCR is based on the principle of DNA amplification by the primer designing and annealing. While QPCR works on the principle of fluorescence probes and dyes. The DNA samples can be detected by using fluorescent probes that emit fluorescence during the DNA amplification or pcr amplification. 

The primer sets are used in the standard polymerase chain reactions while in the QPCR, the primers are labeled with fluorescent dyes and are used in the real-time polymerase chain reaction assay.  The primers used in the standard polymerase chain reaction are not labeled with any probe.

The standard polymerase chain reactions are completed in the three steps: denaturation, annealing, and renaturation or extension.  In QPCR techniques, with these steps, an additional exponential phase of amplification is done to quantify the DNA.

To perform the analysis of the standard PCR, the conventional gel electrophoresis is done using the PCR amplicons.  While in the case of QPCR, the results are recorded by the PCR machine as per the fluorescence emitted by the dyes.

In a standard polymerase chain reaction, the amplicon bands are observed with the help of gel electrophoresis based on the peaks of bands. In the QPCR, different sizes of amplicons are observed in the peaks of QPCR.

PCR Vs RT PCR

In the standard PCR, the electrophoresis is under the pressure of the electrical current, so the amplicon bands migrate towards the positive pole. While in the RT PCR, the fluorescent dye is used for the detection of DNA samples.  The peak in the RT PCR denotes the maximum amplification of DNA samples.

In the above point context, all results of standard PCR are recorded when the process gets completed. But with RT PCR,  with fluorescence, the results are observed soon.

The conventional polymerase chain reaction shows low-resolution amplification, while the RT PCR, shows high-resolution amplification methods for DNA detection.

It is seen that the conventional polymerase chain reaction is a time -consuming which takes almost 3 to 4 hours. While the RT PCR technique takes only a less time of 1 to 1.5 hours to  DNA amplification.