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MM11 - Disease diagnosis molecular biology methods 1 (i) (Gel…
MM11 - Disease diagnosis molecular biology methods 1 (i)
Gel electrophoresis
method of separating + analysing macromolecules (DNA, RNA, proteins)
separation based on size + charge
DNA = protein = -ve, will migrate to anode (+ve)
long fragments move slower than short fragments
fragments placed in gel
polyacrylamide for small fragments
agarose for large fragments
Ethidium bromide stain + UV light make fragments visible
DNA ladder used to measure fragment sizes
Western blotting
detects specific protein in a sample (Qualitative)
also roughly quantitative (gives approx protein quantity)
1) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
SDS keeps protein denatured by disrupting covalent bonds
2) transfer fragments to blot (nitrocellulous membrane)
transfer done through electroblotting
cathode @ gel, anode @ blot, protein migrates to anode (buffer soaked filter paper surrounding)
3) use probe to detect wanted fragment (ELISA)
primary antibody (different species to protein) binds to protein
modified (e.g. chemiluminescent) enzyme-conjugated secondary antibody (different species to 1st antibody) binds to primary antibody
substrate added + secondary antibody releases its detection signal
4) imaging
Nucleic acid hybridisation
use hybridisation probe to attach to + detect specific nucleic acid sequence
stretch of DNA designed to be complementary to the specific sought after sequence
DNA must be ss for hybridisation probe to attach (ds molecule forms)