Polymerase Chain Reaction
PCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA Polymerase to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).
The Cycling Reactions
There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.
1. Denaturation at 94°C :
During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle).
2. Annealing at 54°C :
Large amounts of primers are added to the single strands of DNA.The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded templateThe primers bind to matching sequences along the DNA sequence, in front of the gene that is to be copied. The reaction mixture is then cooled which allows double-stranded DNA to form again. Because of the large amounts of primers, the two strands will always bind to primers, instead of to each other.The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
3. extension at 72°C :
DNA polymerase is added to the mixture. This is an enzyme that makes DNA strands. It can synthesise strands from all the DNA primer combinations and dramatically increases the amount of DNA present. One enzyme used in PCR is called Taq polymerase which originally came from a bacterium that lives in hot springs. It can withstand the high temperature necessary for DNA strand separation and therefore, can be left in the reaction and still functions.
This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and don't give an extension of the fragment.
The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)
This whole process is automated and happens very quickly. The reaction occurs in a small tube which is placed inside a specialised machine which can make the big temperature adjustments quickly.
RT-PCR (Reverse Transcription PCR) is PCR preceded with conversion of sample RNA into cDNA with enzyme reverse transcriptase .
Applications of PCR:
cloning, genetic engineering, sequencing
Limitations of PCR and RT-PCR:
The PCR reaction starts to generate copies of the target sequence exponentially. Only during the exponential phase of the PCR reaction is it possible to extrapolate back to determine the starting quantity of the target sequence contained in the sample. Because of inhibitors of the polymerase reaction found in the sample, reagent limitation, accumulation of pyrophosphate molecules, and self-annealing of the accumulating product, the PCR reaction eventually ceases to amplify target sequence at an exponential rate and a "plateau effect" occurs, making the end point quantification of PCR products unreliable. This is the attribute of PCR that makes Real time quantitative RT-PCR so necessary.
Sources:
Content:
https://archive.innovation.gov.au/Biotechnologyonline/biotec/clonegene.html
https://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml
https://users.ugent.be/~avierstr/principles/pcr.html
Images:
https://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechPCR.shtml