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
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
