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Advanced high school / Biotechnology 1 / 6

Basics of PCR and Restriction-Enzyme DNA Manipulation

The foundation of biotechnology is the technology to cut, amplify, and read DNA freely. Let us understand PCR (Polymerase Chain Reaction) and restriction enzymes, which are central to this technology.

Basic Knowledge

PCR (Polymerase Chain Reaction): A technique for exponentially amplifying extremely small amounts of DNA in a test tube. It was invented by Kary Mullis in 1983, and he received the Nobel Prize in Chemistry in 1993.
The three steps of PCR (repeated in a thermal cycler):
① Denaturation (94°C): Heat separates the DNA double helix into single strands.
② Annealing (50〜65°C): Short single-stranded DNA molecules (primers) complementary to the target sequence bind to both ends.
③ Extension (72°C): Taq polymerase (a heat-stable DNA polymerase from the thermophile Thermus aquaticus) synthesizes complementary strands from the primers.
After 30 cycles, the DNA is amplified approximately one billion-fold (230≒109).

Restriction enzymes (restriction endonucleases): Enzymes that recognize specific base sequences (recognition sequences) and cut double-stranded DNA. They originally functioned as a self-defense mechanism by which bacteria decomposed foreign DNA.
Example: EcoRI recognizes 5'-GAATTC-3' and cuts at the position G↓AATTC. Many such enzymes produce sticky ends, which are used in genetic recombination.

Example Problem: Restriction enzyme EcoRI cuts the recognition sequence GAATTC. When a gene fragment (500 bp) was cut with EcoRI, two bands (300 bp and 200 bp) were obtained. How many EcoRI recognition sequences were present in this fragment? Also state the base sequence of the sticky end.

Answer: Because one cut divides 500 bp into 300 bp + 200 bp, there was one recognition sequence. The sticky end is the four-base overhang 5'-AATT-3' (sticky end).
Key Points
  • PCR = three steps of denaturation → annealing → extension × 30 cycles
  • Taq polymerase = heat-stable and derived from a thermophile
  • Primer = short complementary DNA strand at both ends of the region to be amplified
  • Restriction enzyme = recognizes and cuts a specific sequence (a bacterial defense mechanism)
  • Sticky end = an overhang used in genetic recombination
  • PCR inventor = Kary Mullis (1993 Nobel Prize in Chemistry)

Practice

  1. Explain why a heat-stable DNA polymerase is necessary for PCR.
  2. Explain why restriction enzymes are important in biological experiments using the terms "recognition sequence" and "sticky end."
  3. Theoretically, how many times is DNA amplified after 25 PCR cycles?
Next
Vectors and Genetic Recombination: Cloning Technology

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