PCR is a very efficient method of amplifying a targeted DNA sequence. It is simple test tube method which is easy to perform and inexpensive. It can rapidly amplify a desired sequence of DNA in few hours.This method can be used to amplify any DNA sequences from any source-bacteria,virus,plats or animals.
What are the requirement of a PCR testing?
The standard PCR reaction mixture consists of a large excess of oligonucleotide primer pairs, a template DNA (typically genomic DNA), free deoxynucleotide triphosphates (DNA bases), reaction buffer, and thermostable DNA polymerase (the enzyme that drives the PCR reaction).
What are the steps of PCR?
Primer construction: In the PCR method it is not necessary to know the sequence of the target DNA but we should know the nucleotide sequence of the short segments on the each side of the target DNA.
These stretches of nucleotides are called flanking sequences.
With the help of the flanking sequences 2 oligonucloetide sequences are constructed that are complementary to the respective flanking sequences.
These oligonucloetide sequences complementing the flanking sequence is primer.
Denaturation of DNA:
The DNA to be amplified is heated to seperate the double stranded target DNA into single strands.
Denaturation is done by heating the mixture to 95 degree C.
Annealing:
The mixture is now cooled so that the primers can bind to the flanking sequence.
Chain extension:
The DNA polymerase enzyme in the mixture starts adding the deoxyribonuclotide triphosphates conatining Adenine, Guanine etc to the 3' hydroxyl end of the primer and strand growth occurs towards thr target DNA making complimentary copies of target.
It usually takes 1 min to complete this process and once this process is completed the mixture is reheated so that the process is repeated. About 20-30 such cycles are allowed to run so that there is exponential amplication of target DNA.
What is the type of polymerase?
Thermostable DNA polymerases can withstand temperature of 95°C without denaturation function optimally near 70°C . The standard enzyme currently used in most PCR is derived from the bacterium Thermus (also Thermophilus) aquaticus (Taq).
Advantages:
Rapid
Highly sensitive & specific
Inexpensive
Disadvantages:
Chances of contaimination
Large mutations cannot be detedted.
Primer preparation is difficult
Uses:
Identification of mutation
It can amplify the nucleic acids obtained to large amounts that be easily identified e.g--virus nucleic acids.
Prenatal diagnosis of gene mutation
Forensic analysis of DNA samples
What is RT PCR?
Real-time quantitative PCR (RT-PCR; qPCR) is a highly sensitive method for quantifying the absolute or relative amount of a specific nucleic acid sequence in which the accumulation of PCR products over time is measured directly, without post-PCR modifications.
How can we quantify the desired target DNA?
Quantitation is estimated from the number of cycles of PCR( also know as cycle number or cycle threshold).
In addition to the two primers that are necessary for successful PCR amplification, this method frequently applies a fluorescent probe that hybridizes to the target sequence between the primer pair.
What is the flourescent probe?
In the process of chain elongation the polymerase enzyme adds the nucleotides at its 3' end. Instead of a normal nucleotide, flourescence emitting nucleotides are used. So when 1 cycle of PCR is completed florescence is emitted.
Due to the exponential nature of the PCR, the fluorescence signal increases proportionally to the amount of generated PCR product until a plateau is reached.
Quantification is accomplished by comparing the cycle number at which the patient sample reaches a predetermined level of fluorescence to a standardized curve.
The lower the cycle number higher is the concentration of target DNA or genome in the sample.
For e.g. if the cycle number is low for a viral sample (i.e number of PCR cycles required to emit the standard level of flourescene) higher is the viral load.
What is the advantages of real time PCR?
Rapid test as no post pcr modification is needd.
Highly sensitive
Automated machines with result interpreting softwares are available ,aking the process fast and efficient.
What is reverse transcriptase RT PCR?
All the above examples were of a Double stranded Target DNA.
In cases of single stranded RNA the RNA is first reverse transcribed to form DNA and then the process is taken forward.
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