pcr and its type
TRANSCRIPT
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PCR AND ITS TYPES
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CONTENTS
• PCR Definition
• PCR history
• PCR procedure
• PCR types
• PCR applications
• Conclusion
• References
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• POLYMERASE CHAIN REACTION
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Pcr
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PCR STEPS
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PCR STEPS
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PCR STEPS
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Cycling conditions for amplifying longer PCR products
Step Time/cycles Temperature
Initial activation step 2 min 95°C
3-step cycling
Denaturation 10 s 94°C
Annealing 1 min 50–68°C*
Extension 1 min/kb
Number of cycles 40 cycles 68°C
End of PCR cycling Indefinite 4°C
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PCR
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PCR TYPES
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PCR TYPE
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• Overlap-extension PCR or Splicing by overlap extension (SOEing) :
• Genetic engineering technique
• used to splice together two or more DNA fragments OR complementary sequences.
• It is the technique enables creation of specific and long DNA constructs.
• It can also introduce deletions, insertions or point mutations into a DNA sequence.
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• Nested PCR
• This PCR increases the sensitivity
• Two sets of primers,
• A double process of amplification .
• The first set of primers allow a first amplification. The product of this PCR is subjected to a second PCR using the second set of primers.
• Primers used in the second PCR are specific to an internal amplified sequence in the first PCR. specificity of the first PCR product is verified with the second one.
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• Semi quantitative PCR
• An approximation to the relative amount of nucleic acids present in a sample,
• The markers commonly used are
• Apo A1 and B actin.
• Amplification product is separated by electrophoresis
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• Multiplex PCR
• Multiplex PCR is an adaptation of PCR which allows simultaneous amplification of many
sequences.
• This technique is used for diagnosis of different diseases
• Multiplex PCR can detect different pathogens in a single sample.
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PCR TYPES
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Applications of PCR and impact on science
• PCR and its different variations are highlighted as the most commonly used in laboratories and research institutes.
• Thus, these have contributed to identification
• characterization of several organisms and understanding of physiopathology of diverse
• diseases in human, animal and plants.
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• MEDICINE
• identification of microorganisms
assurance of blood
• Forensic
• As a basic procedure to investigate Deaths
• (paternity testing)
• Evidence from minimal samples of saliva, semen or other tissue debris
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AGRICULTURE
• As conventional PCR or qPCR have also facilitated research in
• Detection of pathogens in plants, animals, and the environment; understanding of their
• Epidemiology and, development of new diagnostic tests, treatments or vaccines.
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• Selective DNA isolation• Isolation of DNA fragments from genomic DNA
by selective amplification of a specific region of DNA.
• This use of PCR augments many methods, such as generating hybridization Probes and DNA cloning which require larger amounts of DNA, representing a specific DNA region.
• PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples Possible.