Full Form of PCR

PCR stands for Polymerase Chain Reaction, which is a molecular biology technique that amplifies a small amount of DNA which helps to make billion copies of specific DNA, used for detailed study to the  scientists. 

PCR is a well known technique in  biological test that help to diagnose the genetic changes in the DNA.  This PCR technique described by Kary Mullins, who worked on oligonucleotides. He got the Nobel prize for this technique in 1969. The PCR was used for detection of  Sickle cell anemia in which mutations occur  in the HBB gene sequence. The enzymatic  amplification occurred in beta globin gene.

Principle of PCR

The polymerase chain reaction is based on the replication of DNA with the help of enzymes DNA polymerases. PCR amplifies the small segment of DNA by primer mediated enzymes. DNA Polymerase helps to synthesize new complementary daughter DNA by Parent DNA.  DNA polymerase  adds new nucleotides in complementary  DNA only on 3’ OH. 

PCR components- using gel electrophoresis

The Polymerase Chain Reaction needs some  components  for making new copies of DNA by replication that initiate and complete the whole process of PCR.

All components is responsible for specific properties such as DNA Template, Taq Polymerase, Buffer system, Primers, Cofactors, Nucleotides triphosphate their working is given below- 

DNA Template

The PCR template helps to make the complementary DNA after heating up on high temperature, two strands separated. This template contains specific target sequences that help to make other strands. 

This DNA sample can be of any DNA source like Genomic DNA (gDNA), Complementary DNA (cDNA), and Plasmid DNA (pDNA). The higher amounts of DNA can cause risk in amplification apart from this lower amount can reduce yields.

The copy  number of DNA can  find out by using Avogadro’s constant and Number of moles ( total mass/ molar mass )of input DNA that can be calculated as-

Copy number of DNA =Avogadro’s Constant * Number of moles 

DNA Polymerase

DNA polymerase is a type of enzyme that helps in replication of DNA by providing new nucleotides and synthesizing new strands of DNA by using Primers in PCR. DNA polymerase adds 60 bases per second, temperature is 70^oC that amplifies the DNA up to 5 kb.

DNA polymerase helps to give better yield even in lower PCR products. A small unit of DNA polymerase is sufficient for amplification of specific DNA.

Primers

Primers are synthetic oligonucleotides, a short single stranded  DNA sequence that is made up of only 10 - 20 bases which attach to a template of DNA. PCR, primers attach on DNA 3’ ends only if their site of attachment is unique or specifically related to target DNA.

The process of inserting Primers in Polymerase chain reaction is-

Buffer system

The  buffer system provides the nutrients or chemical environment to the DNA for amplification and promotes the activity of DNA polymerase. 

It will not allow to change the PH after inserting DNA polymerase and initiate the conditions for denaturation and renaturation.The PH is usually 8.0 - 9.5 that stabilize by HCL (Tris HCL). 

The Components of the Buffer system is Potassium ion that comes from potassium chloride. Magnesium comes from Magnesium chloride MgCl2. Since MG+ and K+ give similar effects to the buffer system. These comoments is important for performing the PCR on optimal conditions.

    Co- solvents and their final concentration for PCR enhancer

Deoxynucleoside Triphosphate (dNTPS)

The dNTPS are a single unit of A, T, C and G which consist of four basic nucleotides - dATPS, dCTPS, dTTP and dGTP. 

These bases are building blocks of new DNA strands. dNTPS are helpful in proofreading so their concentration is important for making complementary DNA, unbalanced concentration of dNTPs are responsible for non- proofreading by  DNA polymerase. 

Uracil glycosylase is a DNA repair enzyme that cleavages uracil containing DNA strands. dUTP is replaced by dTTP that generates uracil PCR products.

Cofactor

During polymerisation, Magnesium ion (Mg2+) works as cofactor. This cofactor catalyzes the phosphodiester bond formed between the 3’ OH and phosphate group. Mg2+ initiates the formation of a complex between the primers and template of DNA to stabilize the negative change on the DNA backbone. 

This cofactor is present as an MgCl2 solution but some polymerase prefer MgSO4 since sulfate helps for performance under circumstances.

PCR Mechanical process

Polymerase chain reaction is completed in three steps: Denaturation and separation , Annealing and Elongation is a repeated cycle. 

1. Denaturation - The sample in the tube is heated at least 94 degrees for only 1- 2 minutes. The heat breaks the Hydrogen bond present between two strands of DNA, changing into single strands used for making another complementary DNA in the next step.

2. Annealing - The second step of PCR, the sample of single strands of DNA cooled at 55- 65^oC. DNA Polymerase and primer add and make 16 - 20 bases length. In this step nucleotides ( A, T, C, G) add and make a complementary DNA. These two stands make in the opposite direction. 

3. Elongation - The last step of PCR in which the sample cooled at 80 degree C. Taq polymerase enzymes end up adding nucleotides in the DNA at 3’ end. DNA polymerase works at optimum condition then adds 1000bp/ minutes. DNA polymerase attach with primers and add other DNA bases, this makes double strands of DNA.

This process is repeated 20 to 70 times, resulting in a complementary DNA sequence that is doubled at every repeated cycle in a very short time period. In a simple, small DNA sequence, separate and make another complementary DNA, one sample amplified again and make billion copies of the sample.

PCR Analysis 

When the PCR is completed, the resulting sequence is analyzed by Electrophoresis. Electrophoresis is a technique used to visualize the fragment of DNA in a gal matrix in the presence of an electric field. The fragments of DNA occur according to their size.The resultant DNA can be visualized under Ethidium Bromide dye. Thick and thin bands make the amount of DNA more or less molecular weight. A band of DNA has many sites of the target region of DNA. PCR is used to visualize enough copies of DNA sequence.

PCR Applications

Polymerase chain Reaction has a vast area in molecular biology. The important application of PCR is identification caused by  microbes in keratitis diseases which cause blindness. PCR is a standard method of identification of infection, more sensitive from other methods like culturing. 

This technique is widely used in various industries like Genetical Engineering, Medical diagnosis, Forensic, Agriculture, sequencing microarray, Paternity testing etc.

1. Genetic Engineering- Polymerase Chain Reaction is to compare the genome of two different organisms for study of the genome. Help to analyze gene expression and Gene mapping. PCR increases the sample of genetic material so this helps to study the  phylogenetic analysis of fossils of DNA. PCR is used to recognize the genetic disorder from fatal DNA of patients. 
2. Forensic -  PCR can amplify the DNA millions or billions times so PCR is helpful for investigation of samples of hair, nails etc. Genetic fingerprinting, DNA testing, genomic typic all testing are based on DNA. So in forensic PCR play an important role in criminal investigations. The PCR helps in DNA paternity test ( Accurate determination of biological father) this helps to identify the victim.
3. Medicine-  In medicine PCR has fields in microbiology, Virology, Mycology , Dentistry.

Microbiology-

• Observation of organism detection.
• Genotyping helps to study Mycobacterium tuberculosis.
• Help to amplify the microorganisms genetic material.

4. Virology-  

•  Help to identify the characteristics of Nucleic acid and their behavior during infection.
• PCR helps in clinical treatment and study about the virus.
• Example- PCR detects the infection of HIV at an early stage.

5. Mycology-

• Diagnose the fungal that causes infection in the body.
• Quick treatment for mycology and parasitology.
• Identify the microorganisms at an early stage that cause infection.