Introduction to Molecular Techniques Flashcards
What is a restriction enzyme?
Produces chiefly by bacteria, cleaves DNA at a specific place, Palindromic: strand has same sequence in diff directions, enables to cut both strand of DNA at same time. Allows for isolation of fragment that contains require gene
What is DNA gel electrophoresis?
-ve DNA travels to +ve electrode, smallest fragments travel furthest. Buffer, power supply. Fluorescent stain, ethidium bromide, visible in UV. Ladder to gauge fragment size. Buffer allows charge on DNA sample across gel
What is the function of PCR and how does it work?
DNA segment amplification tool. Investigate single base mutations, deletions/insertions, variation
Requires forward/reverse primers, taq polymerase, nucleotides.
Temp increased, denaturing, temp reduced, primers form H bonds, temp raised, taq adds nucleotides = 2 copies of target DNA
Describe protein electrophoresis
Proteins = charged molecules, move towards anode/cathode if placed in electric field, dependent on SIZE, SHAPE, CHARGE. Vertical, thinner gel, larger proteins get stuck, thinner move through. Serum protein electrophoresis
Describe SDS-PAGE
Form of protein electrophoresis, separation dependent on SIZE. Adding detergent adds –ve charges, break down disulphide bonds, proteins run in LINEAR shape
Outline isoelectric focusing
Proteins separated based on CHARGE. Tube with high pH at top, low pH at bottom. Proteins migrate up/down tube until they reach pH equal to pI. No net charge at pI so stop migrating
What is 2D-PAGE?
2D electrophoresis. separated by: ISOELECTRIC point and MASS. Good for looking at whole tissue, disease state, can compare normal to abnormal, more protein – is that important to disease, mass spec can identify protein as we know structure/mass
Describe immunoassays
Ab bind specific protein targets (antigens), recognise few epitope: polyclonal/monoclonal. Use western blotting to detect proteins, secondary Ab enzyme linked
What is an ELISA?
antigen coated well, Ab binds, enzyme linked Ab binds, substrate added, converted into coloured product, proportional to amount
Describe gene cloning
restriction enzymes isolate relevant gene, gene put into vector, plasmid, put into suitable host (e.coli), self-replicating ability, gene of interest gets replicated with it = recombination molecule replicate. WHY USE = to produce proinsulin, genetic screening, gene therapy
what is a cathode vs an anode?
Cathode -ve attracts +ve
Anode +ve attracts -ve (DNA)
why use restriction analysis?
investigate size of DNA frag, mutations, variations, clone
What is the difference between poly and monoclonal Ab?
Mono = prod from 1 B cell
Poly = prod from many B cells, recognize many antigens
