Restriction endonucleases cut DNA at specific palindromic sequences, often leaving sticky ends that DNA ligase can join; agarose gel electrophoresis separates the fragments by size.
How it's asked: Naming of EcoRI, palindrome recognition, sticky ends, direction of DNA migration in the gel, and ethidium bromide staining with elution.
Why this is true
Bacteria use restriction enzymes to cut foreign DNA, and each enzyme inspects the length of a DNA molecule until it finds its specific recognition sequence, binds and cuts both strands at particular sugar-phosphate bonds. Recognition sites are palindromes: the sequence reads the same on both strands when read in the same orientation (5′ → 3′). Many enzymes cut a little away from the centre of the palindrome, leaving single-stranded overhangs called sticky ends; any two DNA pieces cut with the same enzyme have complementary sticky ends that hydrogen bond, and DNA ligase then seals the backbone, creating recombinant DNA. Because DNA is negatively charged, fragments placed in agarose gel under an electric field move towards the anode, and the gel's sieving effect makes smaller fragments travel farther.
Key points
Making recombinant DNA
- 1Cut source DNA and vector with the same restriction enzymeComplementary sticky ends
Common traps
- DNA fragments move towards the anode (positive) because DNA is negatively charged.
- In EcoRI, R stands for the strain RY13, not for 'restriction'.
Worked example
After digesting plasmid DNA a student sees three bands on an agarose gel under UV light; the band farthest from the well is needed.
High-yield
EcoRI: E-co-R-I = Escherichia coli RY13, first enzyme. Palindrome GAATTC. Smaller fragments run farther towards the anode; ethidium bromide + UV = orange bands.
Quick check
Q1.In gel electrophoresis, DNA fragments move towards the:
Q2.In the name EcoRI, the letter 'R' denotes: