8.4.3 Investigating the Specificity of Restriction Enzymes

What Are Restriction Enzymes?

• Restriction enzymes are proteins originally discovered in bacteria, where they function to protect against viral DNA by cutting it into fragments.
• Each enzyme recognises a specific DNA sequence (often 4-6 base pairs long) and cuts at or near that site.
• These sequences are often palindromic, meaning the sequence reads the same forwards and backwards on opposite strands.

Investigating Specificity

1. Recognition Sequence:

• Restriction enzymes only cut DNA at their specific recognition site.
• Example: EcoRI recognises the sequence GAATTC and cuts between G and A.

2. Sticky Ends vs. Blunt Ends:

• Enzymes like EcoRI leave sticky ends (single-stranded overhangs), useful for creating recombinant DNA.
• Others, like SmaI, leave blunt ends (no overhangs), which are less efficient for recombination but still useful for specific applications.

3. Experimental Methods to Investigate Specificity:

• DNA is digested with a restriction enzyme and separated using gel electrophoresis to analyse the size of the fragments produced.
• By using DNA with known sequences, scientists can confirm whether the enzyme cuts only at the expected sites.

4. Factors Affecting Specificity:

• Buffer conditions: Specific ions (e.g., magnesium) are required for enzyme activity.
• Temperature: Most restriction enzymes work optimally at 37°C.
• Methylation: Methylation of the recognition site can block the enzyme from cutting.

Applications in Genetic Engineering

• Restriction enzymes are used to:
  • Isolate specific genes from a genome.
  • Prepare DNA fragments for cloning by creating complementary sticky ends.
  • Cut plasmids to insert genes for recombinant DNA production.

Key Terms for Exams

• Restriction enzyme: Protein that cuts DNA at specific sequences.
• Recognition sequence: Specific base sequence recognised and cut by the enzyme.
• Sticky ends: Single-stranded overhangs left after a staggered cut.
• Blunt ends: Straight cuts with no overhangs.
• Gel electrophoresis: A technique used to separate DNA fragments by size.