PCR (Polymerase Chain Reaction) Machine

            PCR stands for "Polymerase Chain Reaction" in biological sciences, especially in molecular biology. A molecular biology technique which makes millions of copies of a target DNA. A target single copy of DNA molecule is exponentially amplified which produces millions of copies of that particular target DNA segment with the help of primers, enzymes and nucleotides.

      PCR has wide range of applications in molecular biology.. This technique has been regularly used in clinical and medical laboratories, in biological or biomedical research and forensic sciences (crime investigation. 

Most of PCR works on the principle of Thermal Cycling. Thermal Cycling is a process where the DNA molecules and constituents along with that segment is repeatedly heated and cooled. 

Principle of PCR: 

       
           "In the first step, the target DNA segment is heated at about 95℃ which will denature the DNA into two single stranded segments, temperature is then cooled so that primers will bind to the single stranded target DNA. Primers and DNA segment then become the templates for the DNA polymerase which will enzymatically initiate the annealing and extension process by assembling the free oligo nucleotides. A copies of the target segment is then generated. This generated copies are then used as a template to generate many more copies of the target DNA segment by repeating the cycles (Denaturing, Annealing and extension). Number of cycles range from 30-40 per PCR Reaction which will generate the many copies of target DNA segment exponentially".

Schematic Representation of PCR Reaction

A PCR reaction tube consists of a target DNA Segment, Primers, Polymerase Enzyme, Free Oligo nucleotides, PCR buffer.

Target DNA Segment: this is a template which will be amplified. The size of the DNA segment can vary from 100 base pairs to 10,000 base pairs.. In some cases, investigators have used up to 40,000 base pairs. 

Primers: these are short segments of DNA which are in the size between 10 base pairs to 20 base pairs. These short segments are complementary to the target DNA segment. They will bind to the complementary sites during annealing step. "The purpose of PCR primers is to provide a “free” 3’-OH group to which the DNA polymerase can add dNTPs".

Polymerase Enzyme: when primers anneal complementary to the target DNA, polymerase enzyme usually Taq Polymerase starts assembling the free oligonucleotides to the target segment  to form the copy of the target DNA segment. 

Oligo Nucleotides: these are free nucleotides which are essential for the DNA and act as the building blocks for the DNA molecule. 

PCR Buffer: buffer usually contains Tris-HCl, KCl and in some cases MgCl2. This buffer is required to maintain the optimal conditions for the activity of the polymerase enzyme. 

How does PCR Machine Work?

            A PCR tube is kept on a PCR Tube stand in pcr machine. PCR tube consists of template DNA, Primers, Taq Polymerase enzyme, PCR buffers, oligo nucleotides and also water to make up the volume.. Total volume ranges from 10µL to 50µL.. Usually, 10-20µL is the best for proper performance of the PCR. 

PCR machine works in three stages. 

Stage 1: In this stage, temperature is raised up to 95℃ for heating which in turn denatures the template double stranded DNA into single stranded DNA.. this step is performed for about 2 to 5 minutes. This is called as denaturing stage. 

Stage 2: In this stage, there are 3 steps involved.

1st step is denaturing step, which is for about 15 to 30 seconds and then temperature is cooled down to 50 to 60℃. 

2nd step is annealing step, where primers will anneal to complementary DNA strands and polymerase enzyme will bind to primer and template strand, this step is usually lasts for 30 to 60 seconds (which allows the enough time for the primers to anneal to the template strands).

Then temperature is raised up to 72℃.

3rd step is extension step, where polymerase enzymatically starts assembling the free oligo nucleotides to the template strand. at 72℃, the polymerase will assemble the free oligonucleotides (100 base pairs per second) and this step usually lasts for up to 1 minute. (This time is depends on the extension requirement of the template strand).

In this manner, these steps are repeated for about 30 to 40 times, which is called as number of cycles. which will eventually generates many copies of the template strand. 

Stage 3: This stage is called as extended extension, where temperature is 72℃ and the polymerase will add the left over oligo nucleotides if necessay to complete the template strands. 

After these 3 stages, temperature is then cooled 4℃, where reaction stops and can be kept hold. 

Applications of the PCR:

* Generating the hybridization probes. 

* DNA Cloning

* DNA Fingerprinting, DNA Sequencing. 

* Recombinant DNA Technology

* Identifying genetic relation between individuals. 

* Gene expression studies. 

* Forensic Science (Crime investigation), genetic fingerprinting. 

* Pre-implanatation Genetic Diagnosis.

* Tissue Typing which is vital to organ transplantation. 

* PCR allows rapid and highly specific diagnosis of infectious diseases, including those caused by the bacteria and viruses. 

* In forensic sciences, pcr is useful for the DNA finger printing, or genetic finger printing which allows the testing of DNA Paternity tests.