Molecular Scissors and Glue

Scissors that read a sequence

To paste a gene somewhere, you first need to cut it out cleanly. A restriction enzyme is a protein, originally borrowed from bacteria, that scans DNA and cuts it only where it finds a specific short sequence — its recognition site. The enzyme EcoRI, for instance, cuts wherever it sees the sequence GAATTC. Because that exact sequence appears only at certain places in a genome, the same enzyme always cuts the same molecule into the same predictable pieces.

Many recognition sites are palindromes — they read the same on both antiparallel strands. GAATTC on the top strand pairs with CTTAAG on the bottom, which read backward is again GAATTC. This symmetry is why one enzyme can cut both strands at matching positions.

Sticky ends: why the magic works

Some restriction enzymes cut the two strands at slightly offset points, leaving a short single-stranded overhang on each fragment. These overhangs are called sticky ends. They are 'sticky' because the overhang is happy to find a partner with the matching, complementary overhang and snap together by base pairing.

EcoRI recognition site (cut offset, leaving sticky ends):

   5'-G     A A T T C-3'
   3'-C T T A A     G-5'
        ^cut here   ^cut here

After cutting, each fragment has a single-stranded AATT overhang:

   5'-G          A A T T C-3'
   3'-C T T A A          G-5'
         (overhang)  (overhang)

Any TWO fragments cut by EcoRI now share the SAME AATT overhang,
so a human-gene fragment and a plasmid fragment can pair up:

   ...G  +  A A T T C...   ->   ...G A A T T C...
   ...C T T A A  +  G...        ...C T T A A G...

The glue: DNA ligase

Sticky ends pairing up is only a temporary embrace — the two fragments are held by a handful of hydrogen bonds, which would slip apart. To make the join permanent, DNA ligase seals the gaps in the backbone, forming the real covalent bonds that turn two loose pieces into one continuous, stable molecule.

1. Cut both the donor gene and the carrier with the same restriction enzyme so they share sticky ends.
2. Mix the two; their complementary overhangs base-pair and the pieces line up.
3. Add DNA ligase to seal the backbone, locking the new recombinant molecule together for good.