Gateway Cloning

Gateway cloning uses the site-specific recombination system of bacteriophage lambda. It eliminates the need for restriction enzymes and DNA ligase by using recombination sequences recognized by specific enzymes.

The system works in two reactions:

• BP reaction: a PCR product flanked by attB sites recombines with a donor vector (pDONR) containing attP sites. The BP clonase enzyme mix catalyzes the reaction, producing an entry clone with the insert flanked by attL sites.
• LR reaction: the entry clone recombines with a destination vector containing attR sites. The LR clonase transfers the insert into the destination vector, which provides the appropriate expression elements (promoter, tag, selection marker) for the target organism.

Gateway cloning is directional, preserves the reading frame, and allows the same entry clone to be shuttled into dozens of destination vectors (e.g., for expression in bacteria, mammalian cells, yeast, or plants). The main limitation is the size of the recombination sites (attB1 and attB2 are ~50 bp each), which add extra amino acids to the expressed protein unless removed by a protease cleavage step.

TOPO Cloning

TOPO cloning uses topoisomerase I from Vaccinia virus, which cleaves DNA and remains covalently bound to the 3′ phosphate. A linearized TOPO vector has topoisomerase attached to both ends. When a PCR product with compatible ends is added, the topoisomerase religates the vector, inserting the PCR product — no ligase needed.

TOPO TA cloning: Taq polymerase adds a single 3′-A overhang to PCR products. The TOPO vector has complementary 3′-T overhangs. The PCR product is mixed with the linearized vector at room temperature for 5 minutes, then transformed into competent cells. It is the simplest cloning method available, with no restriction digestion, ligation, or post-PCR purification required.

TOPO blunt-end cloning: PCR products with blunt ends (from proofreading polymerases) are cloned using a version of the TOPO vector optimized for blunt-end ligation. The efficiency is comparable to TA cloning with the correct vector.

Choosing Between Them

TOPO cloning is faster (5 minutes at room temperature) and better suited for quick subcloning of a single insert. Gateway requires more setup (attB adapters on PCR primers, separate entry and destination vectors) but scales to high-throughput applications and makes swapping between expression systems trivial.