The enzymes of rDNA technology

rDNA technology uses a toolkit of enzymes.

Nucleases cut the phosphodiester bonds that hold nucleic acids together: DNases cut DNA, RNases cut RNA. Exonucleases nibble nucleotides off one at a time from an end; endonucleases cut inside the chain.

Restriction enzymes are endonucleases that cut DNA at a specific recognition site, usually 4-8 base pairs long. Bacteria make them to cut up the DNA of attacking phages. Werner Arber, Daniel Nathans and Hamilton Smith shared the 1978 Nobel Prize for discovering them. There are three types:

• Type I cuts far from its site (about 1000 bp away) and Type III about 24-26 bp away. Both need SAM, Mg²⁺ and ATP.
• Type II cuts inside its recognition site and needs only Mg²⁺. These are the ones used in rDNA technology, because they cut at a predictable place.

Type II sites are palindromes: the sequence reads the same on both strands when each is read 5'→3'. The cut leaves a 5'-phosphate and a 3'-OH on the ends.

• If the two strands are cut at staggered points, the ends have short single-stranded overhangs: sticky ends (as with HindIII and PstI).
• If both are cut at the same point, the ends are blunt (as with EcoRV).

Naming: the genus initial, then two letters of the species, then the strain, then a Roman numeral for the order of discovery. EcoRI comes from Escherichia coli (E + co), strain RY13, the first enzyme found; HindIII from Haemophilus influenzae, strain Rd, the third.

The other tools:

• DNA ligase joins DNA strands. T4 ligase uses ATP and can join sticky or blunt ends; E. coli ligase uses NAD.
• DNA polymerase builds new DNA in the 5'→3' direction, but needs a primer to start.
• Alkaline phosphatase removes 5' phosphates; polynucleotide kinase adds them.
• Terminal transferase adds a tail of nucleotides to 3' ends without a template.
• Reverse transcriptase makes cDNA from an RNA template.
• Poly A polymerase adds a tail of A's to the 3' end of RNA.