Slide 1

Topic: Biotechnology- Principles and Processes - Polymerase Chain Reaction

• Polymerase Chain Reaction (PCR)
• Definition: a laboratory technique used to amplify a specific segment of DNA
• Invented by Kary Mullis in 1983
• Used in various applications, such as genetic testing, gene cloning, and forensics
• PCR can be done in three steps: DNA Denaturation, Annealing, and Extension

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PCR step 1: DNA Denaturation

• DNA double helix is heated to separate the two strands
• Denaturation temperature is typically 94-98°C
• Hydrogen bonds between base pairs are broken
• Two separate DNA strands are formed

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PCR step 2: Annealing

• Temperature is reduced to allow the primers to bind to the DNA template
• Primers are short DNA sequences that are complementary to the target DNA region
• They provide a starting point for DNA synthesis
• Primers bind to the specific DNA sequence of interest

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PCR step 2: Annealing (Contd.)

• Annealing temperature is typically 50-65°C
• Primers attach to the single-stranded DNA template through hydrogen bonding
• Primers bind to the 3’ end of the target DNA sequence in opposite directions

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PCR step 3: Extension

• DNA polymerase synthesizes new DNA strands complementary to the template strands
• Temperature is increased to allow DNA polymerase to work optimally
• Extension temperature is typically 70-75°C
• DNA polymerase adds free nucleotides to the 3’ end of the primers

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PCR step 3: Extension (Contd.)

• DNA synthesis occurs in the 5’ to 3’ direction
• DNA polymerase adds nucleotides to the growing DNA strand
• The DNA strands are replicated exponentially during each cycle of PCR
• Each cycle doubles the amount of target DNA

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Components required for PCR

• DNA template: The DNA sequence to be amplified
• Primers: Short DNA sequences that bind to the target DNA region
• DNA polymerase: Enzyme that synthesizes new DNA strands
• dNTPs: Deoxyribonucleotide triphosphates (A, T, C, G) for DNA synthesis
• Buffer: Provides optimal reaction conditions for DNA polymerase

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Types of PCR

• Real-time PCR: Allows monitoring of the PCR process in real-time
• Reverse transcription PCR (RT-PCR): Converts RNA to complementary DNA (cDNA) for amplification
• Nested PCR: Two rounds of PCR to enhance specificity and sensitivity
• Multiplex PCR: Simultaneous amplification of multiple target DNA sequences

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Applications of PCR

• Genetic testing: Used for identifying genetic disorders or mutations
• Gene cloning: Amplifies and produces large amounts of DNA for further studies
• Forensic analysis: Amplifies DNA from crime scene samples for identification
• Diagnosis of infectious diseases: Detects pathogens in patient samples

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Advantages of PCR

• High sensitivity: Can detect minute amounts of DNA
• Rapid and efficient: Amplification can be completed within hours
• Specific: Primers ensure amplification of target DNA sequence only
• Versatile: Can be used for a wide range of applications
• Cost-effective: Requires minimal equipment and reagents That’s an invalid markdown format. Please use the following format for the requested slides:

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Please provide the slide content in the correct format, and I will be happy to help you create slides 11 to 20. Apologies for the confusion earlier. Here are the slides 21 to 30 on the topic “Biotechnology- Principles and Processes - Polymerase Chain Reaction”:

Slide 21

• PCR Primers:
  • Short DNA sequences
  • Typically 18-25 nucleotides long
  • Designed to be complimentary to the target DNA sequence
  • Important for specificity of amplification
  • Forward and reverse primers used in PCR

Slide 22

• PCR Cycling Parameters:
  • Denaturation: 94-98°C for 30 seconds to 2 minutes
  • Annealing: 50-65°C for 30 seconds to 1 minute
  • Extension: 70-75°C for 1-2 minutes
  • Number of cycles: usually 25-35
  • Parameters can be optimized based on the specific target DNA and primer requirements

Slide 23

• PCR Optimization:
  • Adjusting reaction conditions for optimal results
  • Factors to consider:
    • Primer concentration
    • Mg2+ concentration
    • Annealing temperature
    • Extension time
    • DNA template quality and concentration

Slide 24

• Gel Electrophoresis:
  • Technique used to separate DNA fragments by size
  • DNA samples are loaded onto an agarose gel
  • Applied electric current causes DNA to move towards the positive electrode
  • Smaller DNA fragments move faster and travel further than larger fragments

Slide 25

• Gel Electrophoresis (Contd.):
  • After electrophoresis, DNA fragments can be visualized using a DNA stain (e.g., ethidium bromide)
  • DNA bands can be compared to size markers to determine fragment size
  • Gel electrophoresis is commonly used to analyze PCR products

Slide 26

• Quantitative PCR (qPCR):
  • Also known as real-time PCR
  • Monitors the amplification of DNA in real-time
  • Uses a fluorescent probe that emits fluorescence when bound to the amplified DNA
  • Measures the number of PCR cycles required for the fluorescence to reach a certain threshold

Slide 27

• Reverse Transcription PCR (RT-PCR):
  • Converts RNA to complementary DNA (cDNA) for amplification
  • Utilizes the enzyme reverse transcriptase to synthesize cDNA
  • Allows for the detection and quantification of RNA transcripts
  • Widely used for gene expression analysis

Slide 28

• Nested PCR:
  • Two rounds of PCR amplification
  • First round uses outer primers to amplify a larger DNA fragment
  • Second round uses inner primers that are complementary to the first round PCR product
  • Increases specificity and sensitivity
  • Useful when amplifying low abundant DNA targets

Slide 29

• Multiplex PCR:
  • Simultaneous amplification of multiple DNA targets in a single reaction
  • Uses different sets of primers, each specific to a target DNA sequence
  • Amplified products can be distinguished based on size or specific fluorescent probes
  • Saves time and resources by reducing the number of reactions required

Slide 30

• Applications of PCR:
  • Medical diagnostics: identification of pathogens, genetic disorders, and cancer markers
  • Forensic analysis: DNA profiling for crime scene investigations
  • Agricultural biotechnology: GMO detection and crop improvement
  • Environmental research: assessing biodiversity and monitoring species abundance
  • Evolutionary biology: studying DNA to understand evolutionary relationships

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