FASCINATING APPLICATIONS OF PCR:HOW AMPLIFIED GENOME APPLIED IN LIFE.

ABSTRACT:

In this article, we will discuss about the fascinating applications of PCR. PCR is a technique use to amplify a specific segment of DNA, generating millions of copies from a single DNA molecule. The process involves three main steps: denaturation, annealing, and extension. These steps cycled multiple times to produce exponential amplification of the target DNA. It has versatile applications in various aspects of biological fields. We will also provide references to learn and acknowledge applications of PCR in real life.

INTRODUCTION-APPLICATIONS OF PCR:

PCR has several applications in medical, clinical, industrial, pharmaceutical and research fields. Sex determination, forensic analysis, mutation detection, pharmacogenomics are some examples of real life applications of polymerase chain reaction. Polymerase Chain Reaction (PCR) has revolutionized molecular biology and has become an indispensable tool in various scientific disciplines. Since its inception in the 1980s by Kary Mullis, PCR has undergone numerous advancements and has found diverse applications across fields ranging from genetics and forensics to medicine and environmental science.

APPLICATIONS OF PCR:

1. GENETIC TESTING AND DIAGNOSIS-MODERN APPLICATION OF PCR:

PCR plays a crucial role in genetic testing and diagnosis of various genetic disorders and diseases. It allows for the detection of mutations, polymorphisms, and gene expression patterns associated with diseases such as cystic fibrosis, Huntington’s disease, and cancer.

STEPS:

1. Reverse Transcription of pathogen mRNA
2. Denaturation
3. Primer addition
4. DNA polymerase addition
5. Repetition of steps
6. Detection through gel electrophoresis

2. INFECTIOUS DISEASE DIAGNOSIS:

PCR widely used in the diagnosis of infectious diseases due to its high sensitivity and specificity. It enables the rapid detection of pathogens such as bacteria, viruses, and fungi, aiding in the timely diagnosis and treatment of diseases such as COVID-19, HIV, tuberculosis, and hepatitis.

PROCEDURE TO DETECT INFECTION:

PCR tests work by:
● Obtained a sample of blood, saliva, mucus, or tissue
● Then put it in a special machine. An enzyme called polymerase added to the sample.
● This causes the sample to produce copies.
● The copying process repeated multiple times. After about an hour, billions of copies are
made.
● If a virus or pathogen is present, it will be indicated on the machine.
● Certain viruses, including COVID-19, made up of RNA rather than DNA. For these
viruses, the
● RNA must changed into DNA before copying. This process called reverse transcription
PCR

3. FORENSIC ANALYSIS-MOST ABUNDANT APPLICATION OF PCR:

PCR has become an essential tool in forensic science for DNA profiling and identification. It allows for the analysis of minute DNA samples collected from crime scenes, helping to establish links between suspects and evidence, and to exonerate innocent individuals.

METHODS OF DNA PROFILING:

RFLP based DNA fingerprinting: it consist of following steps:

1. DNA extraction
2. Treatment with restriction enzyme
3. Gel Electrophoresis of fragments
4. Denaturation of fragments
5. Transfer of bands on nylon sheet
6. Addition of specific probes on sheat
7. X ray film analysis

PCR based DNA fingerprinting: it has following steps:

1. DNA sample extraction
2. Amplification of sample DNA
3. Gel Electrophoresis
4. Visualization through laser beam

4. ENVIRONMENTAL MONITORING:

PCR is employed in environmental science for the detection and quantification of microorganisms and pollutants in various environmental samples. It enables the monitoring of water and soil quality, assessment of microbial diversity, and detection of contaminants in food and water supplies.

5. PHARMACOGENOMICS:

PCR facilitates pharmacogenomic studies by identifying genetic variations that influence an individual’s response to drugs. It helps in optimizing drug therapies, predicting drug efficacy and toxicity, and personalizing medicine based on an individual’s genetic makeup.

Toxicity is a major concern in the testing of new potential drugs, and real-time RT-PCR arrays provide
a rapid, comprehensive overview of how cells respond to these compounds. Arikawa and colleagues demonstrated the effectiveness of RT2 Profiler PCR Arrays in distinguishing
between a diabetes drug that was discontinued due to high liver toxicity, troglitazone (Tro). Arikawa treated HepG2 liver cells with each of the drugs, and then studied gene expression using the
Human Stress & Toxicity Pathway Finder PCR Array and the Human Drug Metabolism PCR Array. Both arrays yielded different gene expression profiles for Tro than for Rosi and Pio, demonstrating the
ability of real-time RT-PCR to predict, corroborate, and suggest explanations for clinical observations. In particular, the Human Stress & Toxicity PathwayFinder PCR Array showed significantly higher
expression levels of the genes HSPA6, CRYAB, and CSF2 with Tro treatment. Moreover, gene expression relating to drug metabolism was also altered.

By analyzing gene expression changes, the team was able to demonstrate significant
differences in a cell system model, between a drug that humans can tolerate and a drug with
unsafe toxicity levels.

6. EVOLUTIONARY BIOLOGY-APPLICATIONS OF PCR:

PCR is utilized in evolutionary studies to analyze genetic variation and phylogenetic relationships among different species. It enables researchers to reconstruct evolutionary histories, study population genetics, and trace the origins and spread of species over time.

7. AGRICULTURE AND FOOD SAFETY:

PCR is employed in agriculture for crop improvement, disease resistance breeding, and the detection of genetically modified organisms (GMOs) and foodborne pathogens. It ensures the safety and quality of agricultural products and helps in regulatory compliance. It is done by PCR cloning process.

In PCR cloning, a PCR reaction is performed to amplify the sequence of interest, and then it is joined to the vector via a blunt or single-base overhang ligation prior to transformation. The process involves designing primers for PCR-based cloning, Running the product on a gel to ensure the anticipated size and sufficient DNA, and adding restriction sites to the ends of the DNA fragment for easy cloning into a plasmid of interest.

CONCLUSION-APPLICATIONS OF PCR:

The widespread adoption of PCR across various scientific disciplines underscores its versatility and significance in modern research and diagnostics. As technology continues to advance, PCR techniques are becoming more sensitive, faster, and cost-effective, further expanding its applications and driving innovations in molecular biology and related fields.

REFERENCES:

Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., & Erlich, H. (1986). Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology, 51(0), 263-273. https://pubmed.ncbi.nlm.nih.gov/3472723/

Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., & Arnheim, N. (1985). Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230(4732), 1350-1354. https://pubmed.ncbi.nlm.nih.gov/2999980/

Cheng, S., & Fockler, C. (2007). Applications of the polymerase chain reaction in clinical diagnostics. Methods in Molecular Biology, 429(0), 1-12. https://pubmed.ncbi.nlm.nih.gov/7580841/

Rani, A., & Kumar, S. (2010). Applications of polymerase chain reaction in molecular medicine. Int J Hum Genet, 10(1), 53-58. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768498/

Settin, A. (2008, January 1). Rapid sex determination using PCR technique compared to classic
cytogenetics. PubMed Central (PMC). https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068716/