Polymerase Chain Reaction (PCR) Overview

Polymerase Chain Reaction (PCR)

• PCR is an in-vitro technique for amplifying a section of DNA.
• Amplification means making numerous copies of a DNA segment.
• PCR can create billions of copies of a target DNA sequence in a short time frame.

PCR Outline

• Explain PCR principles and procedures.
• Discuss medical applications of PCR.

PCR Components

• DNA sample: The DNA segment to be amplified.
• Taq polymerase: A heat-stable DNA polymerase that works at high temperatures (up to 95°C).
• dNTPs (deoxynucleotide triphosphates): Nucleotides used to build new DNA strands.
• Primers: Short, single-stranded DNA sequences that are complementary to the target DNA sequence.
  • One primer binds to the 5' end of one DNA strand.
  • The other primer binds to the 3' end of the complementary DNA strand.
• Thermal cycler: An apparatus used to perform repeated cycles of temperature changes.

PCR Cycle Steps

• Denaturation (95°C): The DNA double helix is separated into two single strands.
• Annealing (50-60°C): The primers bind to their complementary sequences on the single DNA strands.
• Extension (72°C): The Taq polymerase extends the primers, adding nucleotides to synthesize new DNA strands from 3'end of the primers.

PCR Thermal Cycler Procedure

• The DNA sample, DNA polymerase, buffer, nucleoside triphosphates, and primers are placed in a thin-walled tube.
• These tubes are put into a thermal cycler.
• The thermal cycler performs the cycles of denaturation, annealing, and extension.

PCR Protocol

• Reagents are placed into a PCR tube.
• The DNA double helix is separated into two single strands (denaturation).
• Primers are bound to their complementary sequences on single strands.
• The polymerase extends the primers, adding nucleotides to form new DNA strands (extension).

PCR Temperature Protocol

• Initial Melt (94°C for 10 minutes).
• Melt (95°C for 30 seconds).
• Anneal (55°C for 30 seconds).
• Extend (72°C for 1 minute).
• Final extension (72°C for 6 minutes).
• Hold (4°C).

PCR Cycle Steps (Detailed)

• Step 1: Denaturation (95°C): DNA strands are separate.
• Step 2: Annealing (55°C): Primers attach to complementary sequences.
• Step 3: Extension (72°C): DNA polymerase adds nucleotides to the 3' ends of the primers.

Gel Electrophoresis of DNA

• Gel electrophoresis separates DNA fragments based on their size (length).
• Smaller DNA fragments move further through the gel than larger fragments.
• Electrophoresis detects the presence of DNA and the number of nucleotides in a DNA fragment.

PCR Types & Variations

• Multiplex PCR
• Asymmetric PCR
• Hot-start PCR
• Inverse PCR
• Nested PCR
• Quantitative PCR (Q-PCR)
• Reverse Transcription PCR (RT-PCR)
• Thermal asymmetric interlaced PCR (TAIL-PCR)
• Touchdown PCR

Advantages of PCR

• Extremely high sensitivity.
• Easy to set up.
• Fast turn-around time.

Disadvantages of PCR

• Extremely liable to contamination.
• Requires high degree of operator skill.
• Positive results can be difficult to interpret (e.g., latent viruses).

Medical Applications of PCR

• Amplification and quantification of DNA and RNA.
• Diagnosis of diseases.
• PCR in comparison of different genomes.
• Drug therapy.
• Diagnosis of genetic disorders like thalassemia and CML (Chronic myeloid leukemia).
• Detection of pathogens (viruses, bacteria, parasites, fungi).
• Gene therapy monitoring.
• Molecular diagnostics.
• Drug development (measuring drug efficacy).
• Cancer detection and treatment.