Fri. Nov 15th, 2019

Real Time PCR (Principle)

4 min read
Real Time PCR is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).  The term "real time" denotes that it can monitor the progress of the amplification when the process is going on. It is used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified.

Real time Polymerase Chain Reaction (Real-Time PCR), also known as quantitative polymerase chain reaction (qPCR), is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR).

It is used to monitor the progress of a PCR reaction in real time. At the same time, a relatively small amount of PCR product (DNA, cDNA or RNA) can be quantified (i.e. It monitors the amplification of a targeted DNA molecule during the process).

The term “real time” denotes that it can monitor the progress of the amplification when the process is going on in contrast to the conventional PCR method where analysis is possible only after the process is completed.

 

Terminologies in Quantative PCR

Amplicon – Short DNA segment obtained from PCR amplification.

 

Baseline– Fluorescent signal level during the initial cycles of PCR. It is used to accurately determine threshold cycle (Ct).

 

Threshold– Level of detection or the point at which a reaction reaches a fluorescent intensity.  It is set to distinguish relevant amplification signal from the background.

 

  • Level of signal that reflects a statistically significant increase over the calculated baseline signal. It is usually set at 10 times the standard deviation of the fluorescent signal of the baseline.

 

Threshold Cycle- Number of cycle at which the fluorescent signal cross the threshold.

  •  Ct (threshold cycle) is the intersection between an amplification curve and a threshold line. It is a relative measure of the concentration of target in the PCR reaction.

 

Dynamic Range– The range over which an increase in starting material on concentration gives rise to a corresponding increase in product.

  • This should be a 7–8 log range for plasmid DNA and at least a 3–4 log range for cDNA or genomic DNA.

 

Slope– The slope of the log–linear phase of the amplification reaction is a measure of reaction efficiency.

  • To obtain accurate and reproducible results, reactions should have an efficiency as close to 100% as possible, equivalent to a slope of −3.32. i.i The slopeof standard curve should be as close as possible of -3.32 (-1/log(2)), meaning that for each PCR cycle, it can perfectly double DNA quantity.

 

Reporter dye- The dyes that binds to double stranded DNA (Amplified products) such as SYBR green.

 

Passive reference dye– A dye that provides an internal reference to which the reporter dye signal can be normalized during data analysis.

  • Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye. Therefore, a lower amount of ROX dye would produce a higher Rn value assuming fluorescence signal from FAM dye is unchanged.

 

NTC (No-Template Control) A sample without DNA template.

 

This technique is commonly used to measure gene expression. It is more sensitive than microarrays in detecting small changes in expression but requires more input RNA and is less adaptable to high-throughput studies.

Real-time PCR uses an increase in the intensity of a fluorescent signal generated by an intercalating dye or from the breakdown of a dye-labeled probe during amplification of a target sequence to detect nucleic acids either for their presence or absence or for their amount.

A PCR reaction needs a pair of primers that are complementary to the sequence of interest. Primers are extended by the DNA polymerase. The copies produced after the extension, so called amplicons, are re-amplified with the same primers leading thus to an exponential amplification of the DNA molecules.

After amplification, gel electrophoresis is used to analyse the amplified PCR products and this in conventional PCR which makes it time consuming; since the reaction must finish before proceeding with the post-PCR analysis. Real Time PCR overcomes this problem.

 

Principle

The principle of amplification used in conventional PCR is employed in real-time PCR. However, the process is monitored in “real-time”. The reaction is placed in to a real-time PCR machine that monitors the progress of reaction with a camera or detector.

A thermostable polymerase enzyme drives a amplification od DNA by synthesizing a complementary sequence of bases to any single strand of DNA providing it has a double stranded starting point.

Although many different techniques are used to monitor the progress of a PCR reaction, they all link the amplification of DNA to the generation of fluorescence which can simply be detected with a camera during each PCR cycle. Hence, as the number of gene copies increases during the reaction, so does the fluorescence, indicating the progress of reaction.

The process can be divided into four stages:

  • Linear ground phase,
  • Early exponential phase,
  • Linear exponential phase (log phase)
  • Plateau phase.

 

In the first phase, PCR is just starting, fluorescent signal has not risen above background. The second phase is where fluorescent signal just rise significantly above background, the cycle at which occurs is called cycle threshold (Ct).

In linear exponential phase, PCR is in its optimal amplification stage with doubling PCR products in every cycle. The last phase is when substrates are exhausted and Taq DNA polymerase is in its end of life, fluorescent signal will no long increase.

Applications

  • In animal and plant breeding, gene copy number is determined using real time PCR.
  • It is used in gene expression analysis such as Cancer Research, Drug Discovery Research.
  • It can also be used in testing of food
  • For disease diagnosis and managementg quantification of virus
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