Biotechnology

Recombinant DNA is DNA assembled from pieces that came from two different sources, so a recombinant molecule by definition carries nucleotides from more than one organism. The technology needs four components, and examiners ask for them as a set: the gene of interest, molecular scissors, molecular glue and an expression system.

The scissors are restriction endonucleases, bacterial enzymes that cut foreign DNA at specific recognition sites as a defence against invading viruses. The cut is staggered rather than square, so each fragment keeps a single-stranded overhang, a sticky end. Sticky ends are the hinge of the subject because they are complementary: a fragment cut with one enzyme will base-pair with a vector cut with the same enzyme, and that pairing is what makes joining possible. Cut vector and insert with two different enzymes and the overhangs no longer match, so nothing joins.

The glue is DNA ligase, which catalyses covalent phosphodiester bonds between the $3'$ hydroxyl of one fragment and the $5'$ phosphate of the next. The third enzyme is reverse transcriptase, which copies RNA into DNA: given mature $m$RNA it yields complementary DNA, or cDNA, and because cDNA carries no introns a eukaryotic gene copied this way can be expressed in a prokaryotic host. A gene for this work can be isolated from a chromosome, synthesised chemically or made from $m$RNA. It cannot be made from protein.