Polymerase Chain Reaction (PCR) Overview

Polymerase Chain Reaction (PCR)

• PCR is an in-vitro technique to amplify a DNA region.
• The sequence of the region is known, or it lies between known sequences.
• PCR amplifies a DNA segment, creating numerous copies
• PCR can produce billions of copies of a DNA sequence in a short period (hours).

Reaction Components

• Reagents Needed
  • DNA sample to be amplified
  • DNA polymerase (Taq polymerase, working at high temperatures up to 95°C)
  • Nucleotides (dNTPs - deoxynucleotide triphosphates)
  • Primer pairs: One primer binds to the 5' end, the other to the 3' end of the DNA strand.
• Apparatus
  • An apparatus to perform cycles of temperature change (e.g. 95°C, 50-60°C, 72°C)
  • 35 cycles are standard

PCR Thermal Cycler

• The DNA sample, DNA polymerase, buffer, nucleotides, and primers are placed in a thin-walled tube.
• These tubes are then placed in a PCR thermal cycler for the amplification process.

PCR Protocol

• Melting/Heat Denaturation:
  • The DNA double helix is separated into two single strands by high temperature (94˚C for 10 minutes initially, and 95°C for 30 seconds).
• Annealing:
  • The temperature is decreased (55°C for 30 seconds) to allow primers to bind to their complementary sequences on the single strands of DNA. 
• Extending:
  • The temperature is raised (72°C for 1 minute) allowing DNA polymerase to add nucleotides to the 3' ends of the primers. This creates new DNA strands by using the old strands as a template. There's also a final extension step (6 mins at 72˚C) to make sure all ends are completed.

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

The Three Main Steps of PCR

• Step 1: Denaturing: At 95°C, DNA is separated into two single strands.
• Step 2: Annealing: At 40-65°C, primers bind to their complementary sequences on the single strands.
• Step 3: Extension: At 72°C, DNA polymerase extends the DNA chain by adding nucleotides to the 3' ends of the primers, creating new strands.

Gel Electrophoresis of DNA

• Gel electrophoresis separates DNA fragments based on size, detecting their presence and quantity in a sample.
• Longer fragments move slower, shorter fragments faster through a gel medium.
•  It analyzes the amplified DNA region from PCR (analyzing the number of nucleotides).

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 (Step-down PCR)

Advantages of PCR

• Extremely high sensitivity (can detect single viral genomes).
• Easy to set up.
• Fast turnaround time.

Disadvantages of PCR

• Liable to contamination.
• Requires high operator skill.
• Interpreting positive results can be difficult (e.g., in cases of latent viruses like CMV).

Medical Applications of PCR

• Amplification and quantification of DNA and RNA.
• Diagnosis of various diseases (including genetic disorders like thalassemia and CML).
• Diagnosis of infectious diseases caused by pathogens.
• PCR for molecular diagnostics and biochemical analyses.
• Drug development and research.

This quiz provides an overview of the Polymerase Chain Reaction (PCR) technique, detailing the components and apparatus required for DNA amplification. It covers the reagents needed, the function of the thermal cycler, and the standard protocol followed during PCR. Perfect for students and professionals looking to understand the fundamentals of DNA amplification.