Making recombinant DNA

Making recombinant DNASpec 8.18

Recombinant DNA is DNA made by joining DNA from two different sources, usually from different organisms. A gene from one organism is combined with a vector, usually a plasmid (a small circular DNA molecule from bacteria), which carries the gene into a host cell.

Getting the gene

• Using reverse transcriptase: mRNA for the gene is extracted from cells that make a lot of it (for example, insulin mRNA from pancreatic beta cells). Reverse transcriptase makes a complementary DNA (cDNA) copy, and DNA polymerase makes it double-stranded. cDNA has no introns, so a bacterium can express it.
• Using restriction endonucleases to cut the gene out of the genome.
• Synthesising the gene from its known base sequence in a gene machine.

Joining the gene to the plasmid

1. A restriction endonuclease (restriction enzyme) cuts DNA at a specific base sequence called its recognition site (often a palindromic sequence). It hydrolyses the phosphodiester bonds in each strand.
2. Many restriction enzymes make staggered cuts, leaving short single-stranded ends called sticky ends.
3. The plasmid is cut with the same restriction enzyme, so its sticky ends are complementary to those on the gene.
4. The gene and plasmid are mixed. The sticky ends join by hydrogen bonds between complementary bases (annealing).
5. DNA ligase forms phosphodiester bonds between the sugar and phosphate groups of adjacent nucleotides (a condensation reaction), sealing the gaps in the backbone to make a recombinant plasmid.

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Common mistake:

Restriction enzymes and ligase have opposite roles: restriction enzymes cut (hydrolysis) and ligase joins (condensation). The sticky ends are held by hydrogen bonds between bases; ligase makes the covalent bonds.