Real Time PCR is a very sensitive and powerful DNA analysis tool. It monitors the amplification of a targeted DNA molecule during the PCR. The principle of the qPCR is same as that of conventional PCR.
Process of qPCR
The working procedure of Real Time PCR can be divided in two steps:
A.Amplification– In this process the Target DNA that is to be studies is amplified or multiplied into number of copies with the help of thermostable DNA polymerase based on PCR principle. The steps in amplification process involves:
High temperature incubation is used to melt double- stranded DNA into single strands and loosen secondary structure in single-stranded DNA. The high temperature employed breaks the weak hydrogen bonds between the purine and pyrimidine molecules leading to formation of single strand.
The highest temperature that the DNA polymerase can withstand is typically used (usually 95°C). The denaturation time can be increased if template GC content is high.
This step involves cooling down the reaction mixture (54-60)°C during which primers bind (anneal) to their complementary sequence in the template DNA.
During annealing, complementary sequences have an opportunity to hybridize, so an appropriate temperature is used that is based on the calculated melting temperature (Tm) of the primers (5°C below the Tm of the primer).
This step usually occurs at 72-80°C (most commonly 72°C). At 70-72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second.
When an amplicon in real-time PCR is small, this step is often combined with the annealing step using 60°C as the temperature.
In this step, the polymerase enzyme sequentially adds bases to the 3′ of each primer, extending the DNA sequence in the 5′ to 3′ direction. Under optimal conditions, DNA polymerase will add about 1,000 bp/minute.
There are two detection methods of Real Time PCR, the first is based on sequence-specific probe such as TaqMan probe, molecular beacon and the second is based on generic non-sequence-specific double-stranded DNA-binding dye such as SYBR green.
The detection is based on fluorescence technology. The specimen is first kept in proper well and subjected to thermal cycle like in the normal PCR. The machine, however, in the Real Time PCR is subjected to tungsten or halogen source that lead to fluoresce the marker added to the sample and the signal is amplified with the amplification of copy number of sample DNA. The emitted signal is detected by a detector and sent to computer after conversion into digital signal that is displayed on screen. The signal can be detected when it comes up the threshold level (lowest detection level of the detector).
Advantages of Real Time PCR
Real Time PCR has many advantages:
- It gives a look in to the reaction that is help to decide which reactions have worked well and which have failed.
- The efficiency of the reaction can be precisely calculated.
- Gel electrophoresis is not required after amplification process as melt curve analysis serve the purpose.
- The real-time PCR data can be used to perform truly quantitative analysis of gene expression while conventional PCR can only give semi-quantitative analysis.
- Faster and less complexity at the quantification of sample.
Thus, qPCR allows the success of multiple PCR reaction to be determined automatically after only a few cycles, without separate analysis of each reaction, and avoids the problem of “false negatives”.