Botany · Ch 4 — Principles and Processes of Biotechnology
Screening for Recombinants
Screening for Recombinants
Successfully getting recombinant DNA into a batch of host cells is only half the job - most cells in that batch will not actually have taken up the vector at all, and even among those that did, only some will carry a genuine recombinant (insert-bearing) copy rather than a self-ligated, empty vector. Screening is the essential step of sorting all of this out: telling which cells are genuine recombinants, expressing the characters the inserted gene confers, apart from the much larger population of non-recombinants that express nothing of the sort. This is a long section because it is really a toolkit chapter in miniature, working through, in turn: blue-white colony selection, antibiotic-resistance-based selection, replica plating, gel electrophoresis (for actually visualising and sizing DNA), the blotting/hybridisation techniques used to identify specific sequences, bioassay of what an inserted target gene actually does, whole-genome sequencing and plant genome projects, DNA-based as …
Differences between Blotting Techniques
Southern, Northern and Western blotting share an almost identical physical workflow - transfer onto a membrane, then hybridisation-based detection - which makes it easy to blur them together, so it is worth being explicit about exactly what differs between the three. Only Southern blotting is literally named after a real person (E.M. Southern); 'Northern' and 'Western' are deliberate plays on that name and describe no actual geography or inventor. They separate three completely different classes of molecule - DNA, RNA and protein respectively - which is really the one fact that determines almost everything else about each technique. Southern and Western blotting both require a denaturation step (unwinding double-stranded DNA, or unfolding protein, respectively) before transfer, while Northern blotting does not. The membrane itself differs too: Southern and Western both typically use nitrocellulose or nylon, while Northern blotting specifically needs amino-benzyloxymethyl paper because RNA does not adhere well to plain nitrocellulose. The nature of the hybridisation/binding event is different in each case - DNA-DNA for Southern, RNA-DNA for Northern, and protein-antibody (not a nucleic-acid hybridisation at all) for Western. And finally, visualisation differs: Southern a …
| Feature | Southern blotting | Northern blotting | Western blotting |
|---|---|---|---|
| Name | Named after the inventor, E.M. Southern | 'Northern' - a play on Southern's name (a misnomer) | 'Western' - likewise a misnomer |
| Separation of | DNA | RNA | Proteins |
| Denaturation | Needed | Not needed | Needed |
| Membrane | Nitrocellulose / nylon | Amino benzyloxymethyl paper | Nitrocellulose |
| Hybridisation | DNA-DNA | RNA-DNA | Protein-antibody |