PCR/ Polymerase Chain Reaction

PCR/ Polymerase Chain Reaction

The Polymerase Chain Reaction one of the techniques most frequently used in molecular biology in order to amplify in vitro a well-defined segment of DNA and of a defined length, it was introduced in 1985 by Kary Mullis. 

Since then, PCR has become an integral part of molecular biology, with applications ranging from basic research to the diagnosis of diseases in clinical medicine and even in research laboratories. To the PCR in biology is used in variable agricultural tests and forensic surveys.

The principle and the experimental conditions which result from it are very simple. It involves carrying out a succession of replication reactions of a double stranded DNA template.

Steps of the PCR

Denaturation

 In order to separate the double stranded of DNA by the heat around 95⸰C.

Annealing

the temperature is lowered to 50-65⸰C to target the amplification on the desired DNA region allowding to  which short DNA molecules called specific PCR primers to bind to the target DNA.

Polymerization

The temperature is increased to 68-72⸰C, so that the DNA polymerase extends the 3 'end of each primer along the template strands or in another way, is a step of polymerization of the strand complementary.

At the end of each cycle, double stranded DNA is synthesized. These steps are repeated or cycled 25 to 35 times to produce exponentially exact copies of the target DNA.

The necessary elements for PCR

• DNA template serves as a matrix for the amplification
• DNA polymerase the most used enzyme is Taq polymerase, extracted from the bacterium Thermus aquaticus.
• Primers that delimit DNA.
• Deoxynucleoside triphosphates (dNTPs)

 PCR Thermocyclers

 They are allowing the process to be automated and a number of thermocyclers are available from different manufacturers.

The versatility of PCR has made it one of the most widely used methods in molecular diagnosis. The number of PCR-based applications has continued to increase rapidly; we attempt to present some of the most important clinical applications of PCR.

The real-time polymerase chain reaction (RT-PCR)

A real-time polymerase chain reaction (real-time PCR), known also as quantitative polymerase chain, is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR) is the continuous collection of the florescence signal from one or more polymerase chain reaction over a range of cycles, and has a crucial role in medical science and biomaterial fields.

Quantitative real time PCR is a sensitive in vitro method that allowed the conversion of fluorescent signal from each reaction into a numerical value for each sample. It is used for many different purposes, particularly for quantifying nucleic acids and for genotyping.

Real-time PCR is used for absolute and relative quantifications of DNA and RNA template molecules and for genotyping in a variety of applications. This includes the determination of viral loads, gene expression, germs and contaminations in food, blood, other body fluids and tissues, allele imbalances and the degrees of amplification and deletion of genes.  Real-time PCR is also becoming increasingly important in the diagnosis molecular biology.

The Reverse transcription PCR (RT-PCR)

uses mRNA as a srating template, the enzyme reverse transcriptase uses the mARN to produce a complementary single stranded DNA in a perocess called reverse transcreption