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.