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samenvatting celmobi: Hoofdstuk 13: DNA-replicatie in vitro en sequentie-analyse
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samenvatting celmobi: Hoofdstuk 13: DNA-replicatie in vitro en sequentie-analyse
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Hoofdstuk 13: DNA-replicatie in vitro ensequentie-analyse
• Polymerase chain reaction: PCR => exponentiële amplificatie
⤷ Met thermocycler
→ Template, primers (langs beide uiteinden 1)
⤷ bepaalt wat te vermenigvuldigen is
→ TaqI-polymerase (< thermofiele bacteriën)
→ Denaturatie = scheiden v/d strengen door temperatuur (95°C)
→ Primers annealing = primers basenparen met template (45°C – 60°C)
→ Extensie: DNA-synthese vanaf primer (72°C)
→ Herhaal 30x
→ Hypervariabele sequenties! → VNTR die verschillende # hebben van individu tot individu
⤷ ”microsatellieten”
= Short tandem repeats (STR)
= Simple sequence repeat (SSR)
• Gevoeligheid: contaminatie => vals positief
• Dideoximethode van Sanger: sequentie-analyse
→ Basenparen v/e primer op enkelstrengig DNA
→ 4 verschillende proefbuizen met verschillende dNTP’s en ook met ddNTP
(=ketenterminerend)
⤷ Geen OH’-groep aan de 3’-kant
→ Allemaal verschillende lengtes, maar we weten met welke ddNTP het eindigt => scheiding
op lengte
→ Fluorescentiemarkers voor de 4 ddNTP’s => meer 1 buisje nodig => basendetectie
• Next generation sequencing: methodes die grote # sequenties tegelijk doen
• Nanopore sequencing: DNA door nanoporie en basen o.b.v. elektriciteitsverschillen bepaald
• NIPT (=niet-invasieve, prenatale test): analyse op DNA foetus
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