The Tools - Fine Points

Restriction enzymes

  • Nomenclature (EcoRI): E = genus (Escherichia), co = species (coli), R = strain, I = order of isolation. (First letter genus, next two species.)
  • They are endonucleases (cut internally), not exonucleases (which chew from the ends).
  • They recognise a palindrome - reads the same 5' to 3' on both strands. EcoRI cuts between G and A (G^AATTC), leaving 5' overhangs - sticky ends - that pair with any other fragment cut by the same enzyme and are sealed by DNA ligase. (Some enzymes cut straight across, giving blunt ends.)

Vectors and insertional inactivation

  • A good vector needs an ori (controls copy number), a selectable marker (antibiotic resistance) and a single restriction/cloning site.
  • In pBR322: the ampicillin-resistance gene contains the PstI site, and the tetracycline-resistance gene contains the BamHI (and SalI) site. Inserting DNA at PstI kills ampicillin resistance; inserting at BamHI kills tetracycline resistance - the recombinant is spotted because it has lost that resistance.
  • Blue-white selection: insert into the beta-galactosidase gene; recombinants cannot make the enzyme, so on a chromogenic substrate the recombinant colonies are white and non-recombinants blue.
  • Plant vector = Ti plasmid of Agrobacterium tumefaciens; animal vector = retrovirus (both disarmed).

The three enzyme jobs

Restriction endonuclease (cuts), DNA ligase (joins), DNA polymerase (synthesises/amplifies). 'DNA mutase' and 'DNA recombinase' are not standard cloning tools.

Traps: EcoRI cut = G^AATTC; PstI -> amp, BamHI -> tet in pBR322; recombinant = the colony that has LOST a resistance; probe is NOT a vector (BAC, YAC, plasmid are).

The Process - Sequence and Fine Distinctions

The ordered workflow

Isolate DNA (lysozyme/cellulase/chitinase to break walls; RNase + protease to purify; chilled ethanol to precipitate) -> cut with a restriction enzyme -> separate the fragments by gel electrophoresis (and elute) -> amplify by PCR -> ligate the gene into a vector -> transform the competent host -> select the recombinants -> culture (bioreactor) -> downstream processing.

Gel electrophoresis

DNA is negatively charged, so it moves to the anode (+); smaller fragments travel farther. Bands are seen as orange with ethidium bromide + UV; the band is recovered by elution. (A chromogenic substrate gives BLUE colonies, not DNA bands - a classic trap.)

PCR

Order: denaturation (94 C) -> annealing (primers bind) -> extension. The polymerase must be thermostable (Taq from Thermus aquaticus) so it survives the repeated denaturation. After n cycles there are about 2^n copies.

Fine distinctions

  • Competent host - cells made able to take up DNA by Ca2+ + heat shock; alternatives = micro-injection (animal nucleus) and gene gun / biolistics (gold or tungsten particles, for plants).
  • Cell-wall enzymes by organism: lysozyme = bacteria, cellulase = plant, chitinase = fungus.
  • DNA precipitates in chilled ethanol (isopropanol/room-temperature alcohols are the distractors).

Assertion-Reason watch-outs: PCR needs a thermostable polymerase because the DNA is repeatedly denatured at high temperature (R explains A); a restriction enzyme leaves sticky ends precisely because it cuts off-centre in the palindrome, giving complementary single-stranded overhangs.