Applications of pcr

This presentation include these contents:
What is PCR?
Applications of PCR
Advantages of PCR
Limitations of PCR
PCR vs Cloning
Restrictions of PCR
Things to try if PCR does not work..
Conclusion

1. Topic: APPLICATion of Polymerase chain reaction Mubaika seher bsf1800548 bs zoology (6 th ) Morning BIOLogical Techniques

2. What is PCR? Applications of PCR Advantages of PCR Limitations of PCR PCR vs Cloning Restrictions of PCR Things to try if PCR does not work.. Conclusion Contents

3. “Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence”. POLYMerase chain reaction

4. Advantages of PCR Quick Reliable Sensitive Relatively easy Specific Useful non-invasive procedure Simplicity of the procedure Increased ability to detect less common organisms such as viruses

5. Advantages of PCR Cost-effective Shown to be more cost-effective with selective use than culture and staining Quickly performed in 4-8 hours

6. Disadvantages of PCR Need for equipment Taq polymerase is expensive Contamination False reactions Internal control Cross-reaction Capacity building needed Unspecific amplification Potentially lower specificity compared to culture and staining Supply costs, machinery fees, training expenses

7. Cont. Error rate during amplification Sensitivity to inhibitors Setting up and Running requires high technical skill High Sterile environment should be provided Infidelity of DNA replication. Taq Pol – no Proof reading mech – Error 40% after 20 cycles

8. Applications of PCR PCR in Molecular biology C linical diagnosis DNA sequencing Forsenic Medicine Gen e manipulation and expression studies C omparative study of genomics C omparison with gene cloning Environmental biology Anthropology Mycology and parasitology Animal research S ex determination

9. Application of PCR in molecular biology Used in molecular biology and genetic disease research to identify new genes; for example, the sample containing pathogenic DNA can be PCR amplified using different known specific primers. The amplification indicates presence of pathogenic DNA.

10. In Anthropology In fields such as anthropology and evolution, sequences of degraded ancient DNAs can be tracked after PCR amplification .

11. In Forensic Science With its exquisite sensitivity and high selectivity, PCR has been used for wartime human identification and validated in crime labs for mixed-sample forensic casework With the advent of PCR-based DNA fingerprinting, PCR became an invaluable tool in forensic investigations. Using DNA fingerprinting, tiny fragments of DNA can be isolated from a crime scene and compared to a huge database of DNA of convicts or criminals. It is also useful in ruling out suspects as part of an investigation. DNA fingerprinting is also used in paternity testing, where the DNA from an individual is matched with that of his possible children, siblings, or parents.

12. Mycology and Parasitology PCR technology has also found applications in mycology and parasitology, by enabling early identification of the microorganisms, thus aiding efficient diagnosis and treatment of fungal and parasitic infections.

13. PCR Diagnostics Viruses HIV, SARS, H5N1 PCR can detect the COVID viruses Bacteria meningococcus , legionellosis Analysis for resistant genes MRSA, VRE

14. PCR -In Medical Diagnosis Polymerase chain reaction  ( PCR ) is a broadly applied laboratory test for the  diagnosis  of a wide variety of central nervous system (CNS) diseases, including genetic autoimmune diseases , malignant neoplasms, infections.

15. Diagnosis of Malignant Diseases\Cancer PCR permits early diagnosis of malignant diseases such as leukemia and lymphomas . PCR assays can be performed directly on genomic DNA samples to detect translocation specific malignant cells, infectious agents, like mycobacterium, anaerobic bacteria, or viruses Breast cancer, cervical cancer, chronic myeloid leukemia and other related cancer is detected using different PCR assays. Further, the amount of the mutant gene or oncogene can also be measured . Even the MRD (minimal residual disease) can also be determined by the quantitative PCR. Interestingly , PCR is also used in cancer therapy monitoring .

16. PCR –For site Directed Mutagenesis Inserting a mutation in a DNA sequence (called artificial mutagenesis or site-directed mutagenesis) can be useful in removing restriction sites during gene transfer experiments.  This technique is used for introduction of mutations at the desired place in a DNA sequence.

17. Environmental Microbiology The PCR technique has been successfully used to explore many issues in environmental microbiology. Some of its environmental applications are listed below: Sensitive detection of degrading microorganisms in toxic waste and pollutants can be achieved using PCR, which helps efficient biodegradation and bioremediation at the polluted sites. A gene probe-based PCR method has been developed by researchers for the detection of indicator bacteria such as coliforms in water supplies, thus supporting measures that enhance water safety. PCR is also used to detect and monitor water-borne microbial pathogens, which pose a major public health hazard.

18. Sex determination Sex determination can be done accurately using the PCR. A Y chromosome-specific marker is selected for it and amplified using a routine PCR protocol. If amplification is observed, the fetus is male and if amplification is not observed the fetus is female. Moreover, some X chromosome-specific markers are also used . Genotyping is also used for sex determination of embryos as well as detecting chromosomal and genetic disorders in the foetus .

19. PCR in animal research We are using PCR not only in human and plant research and disease studies but also it is very important for animal research and disease studies. Inherited and infectious animal diseases are being diagnosed using the present method. Some of the common applications of PCR in animal genetics are:  In the identification of MTM mutated gene in dogs, responsible for X-linked Myotubular myopathy. For Bursal disease virus in avian samples.  Identification of canine parvovirus in dogs.  Deletion study of  Meq  gene in chickens

20. Restrictions Of PCR Contamination of reagents or lab results in false positive results Failure due to a mistake in the protocol Different materials/parts of the sample can inhibit the PRC process

21. PCR vs Cloning PCR PCR  enables scientists to produce billions of copies of a piece of DNA within hour.  PCR replicates DNA in an in vitro solution, free of living cells Maximum 4 hours enough for an experiment The amplified DNA is put into many uses because of less error possibility. A nanogram DNA is enough for amplification Automation is present Skilled labour is not required . Less expensive Cloning Cloning  is simply making one living organism from another, creating two organisms with the same exact gene Molecular cloning replicates DNA within in a living cell , 2-4 days should be for an experiment. The amplified of DNA is put into limited number of uses. At a microgram quantity of DNA is required for amplification . Automation is absent Skilled labour is required Highly expensive

22. PCR Cloning Requirement Restriction enzymes DNA ligase Vector DNA Bacterial cells Requirement Taq DNA polymerase RNA primer Free deoxyriboneucleotides are required along DNA segment to be amplified

23. Things to try if PCR does not work.. If no product(of correct size )produced: Check DNA quality Reduce annealing temperature Increase Mg concentration Add DMSO to assay Use different thermostable enzyme Throw out primers –make new stock

24. Investigation strategies and methods Developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from: European Program for Intervention Epidemiology Training Canadian Field Epidemiology Program Thailand Ministry of Health Instituet Pasteur

25. Conclusion PCR  is a highly accurate and rapid method for duplicating genetic material. The discovery of thermostable polymerase enzymes has permitted the automation of  PCR , thus reducing the manpower required to conduct these experiments . With the advent of qPCR, amplified products may also be quantified accurately.