condensed succinct notes using the markschemes and common exam questions. Complete with my own diagrams and flowcharts to help further understand the concept.
In bulletpoint form
Gene therapy eg for CF (cystic fibrosis)
MOLECULAR BIOLOGY - CF caused by mutation
Plasmids suitable - Of CFTR gene
- Small ∴ easily taken up - ∴ prot defective
- Circular - ∴ insert normal, dominant allele
- Markers- easy identify - → DNA
- Easy manipulate - Cells of resp. System
- Cut sp location by rest. Endonuc. - Vector = virus
- Take as spray
Synthesis human insulin by bacteria - Liposomes
1. Isolate mRNA coding for insulin - Harmless virus
2. From B cells pancreas - Unpleasant side effects
3. Reverse transcriptase enz - Short lived effects
4. → cDNA single strand Gel electrophoresis in gen fingerprint
5. DNA p. → double strand 1. Identify DNA + ↑ quantity w PCR
6. Restriction endonuclease (r.end) 2. Cut DNA frags by restriction endo.
7. Sticky ends created 3. Close to VNTR seq
8. Plasmid vector 4. Loaded → wells agarose gel
9. R.end cut plasmid (w micropipette)
10. DNA ligase join DNA 5. Add buffer solution
11. Vector → into host cell 6. Apply direct current
12. Identify modified bact w markers 7. DNA=negative due phosphate group
13. Clone + grow fermenter 8. ∴ DNA attracted anode
9. Short fragments move further
+ves genetic screening 10. Southern blotting
- Provide info abt ↑ed risk have gen 11. Heat to sep strands
cond 12. Fluorescent dye to identify
- Allow prepare late onset conditions 13. UV light
eg huntington 14. Banding pattern
- Allow parents prepare financially Pcr process
- Identify carriers 1. Amplification of DNA
- Help prov early diagnosis 2. = Rapid
- Allow couples both carriers make 3. Only need small sample DNA
decision abt start family 4. DNA denat. 95°
Role genetic counsellor 5. Add DNA primer
- Carriers gen disease 6. Anneal @ 65°
- Gen screening 7. Comp b.p
- Eg amniocentesis 8. Taq p. = ↑ opt t
- Explain test results 9. Rep strand @ 72°
- Discuss Termination 10. Repeat process
- Financial implications 11. Taq doesn’t need replace each cycle
∴ efficient process
, PROK - LAC OPERON
E.g Gibberellin
Operon : length DNA more than 1 struct - T.F = PIF bind promotor reg
gene + control reg DNA (dormant seed)
- ∴ rna p ca bind → initiate transcrp
3 struct genes : - DELLA prot = inhibitor bound PIF
- Lac Z - code B-galactosidase - ∴ x bind gene promote , stop prod
- Lac Y - code permease mrna
- Lac A - code transacetylase - Gibberellin stim breakdown DELLA
prot
When NO lactose present (gene → active) - TF bind target promote : ↑ mrna
- Regulatory gene code for repressor transc amylase gene, ↑ amylase
protein synthesis gene
- Repressor bind operator
- Presence repressor protein , MICROARRAYS
RNA p CAN’T bind to promotor
- NO transcription of 3 struct genes 1. Identify, + compare genes present in
2 species
Lactose present: - DNA collected, cut → frag +
(act as inducer) denatured → s.s (oligo) DNA
- Lactose taken up by bact - DNA labelled fluorescent tags
- Lactose bind allosteric ∴ prev bind - Mix samples allow hybridize comp w
operator probe on microarray
- Transcription x longer inhibited - Any DNA x bind = washed off
- ∴ mRNA transrived - UV identify tags
- Genes translated → synth enz - Scan + store data
EUK - TRANSCRIPTION FACTORS 2. Identify gene expression
(bind rna p, bind t.f, bind sp dna seq) - Isolate collect mRNA from 2 cell
types
E.g OESTROGEN (animals) - Reverse transcriptase ∴ mRNA →
- Diff across c.memb → cytoplasm s.s. cDNA
- Comb w site transcriptional factor - cDNA labelled w fluores.tags +
(already have inhibitor mol) denatured → s.s dna
- T.f >1 site ∴ bind oestrogen - DNA hybridize w probes
- Change shape ∴ release inhib. - Fluoresce = genes being transcribed
- Expose DNA bind site, t.f bind - Intensity → activity gene
- → activate transcription
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