Summary
Samenvatting Methoden in het Biomedisch Onderzoek 3
- Course
- Institution
een samenvatting van alle hoorcolleges en notities. Door dit document te leren haalde ik in eerste zit een 19/20.
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By: aaronverbeek • 8 months ago
By: astridhindryckxprive • 1 year ago
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H1: DNA1. DNA sequentiebepaling
= volgorde van basen in DNA aflezen
a. Toepassingen
De novo genoom sequentiebepaling
- Deels of volledig
- Onbekend genoom van nieuwe organismen sequencen
Resequencing
- Versus referentiegenoom
- Mutaties zoeken
- SNPs zoeken
- Diagnose, farmacogenetica, NIPT…
Aantal DNA/ RNA moleculen tellen
b. 1e generatie
Sanger sequencing
1 primer => 1 streng per keer aflezen
2 aparte reactie = FW en RV primer
betrouwbaarheid verhogen
- korte lengte
- moeite met herhalingen
- lage trouhhput, hoge kosten
Vroeger: 4 versch reacties met 1 ddNTP – scheiden op PAA
gel – aflezen
Nu: fluo gelabelde ddNTPs – scheiding via capillaire
gelelektroforese – aflezen door laser en sensor –
elektroferogram
Phred score => kwaliteitscore
- Q = -10*log(P)
- P = probaliteit van foutieve base call
- Hoge Q = goed
Insertie/ deletie => gemengd signaal > bulk sequencing
1
, Referentiesequentie Human genome project
mens maken Hierarchical shotgun sequencing
Sanger sequencing
Nature 2001
Human Genome sequencing by Celera
Whole genome shotgun sequencing
Sanger sequencing
Geen klonering => mapping moeilijk
Science 2001
Technologische ontwikkeling sequencing
- Automatisering
- Aanpak verandert
Ontwikkeling bio-informatica
Visie op data = open science
Biologische kennis
- Sequentie van genen
- Inzicht evolutie en conservatie
- Genetische verschillen tussen mensen
Nieuwe uitdagingen
- Volledige sequentie
- Meerdere species
- Individuele humane sequenties
- Persoonlijke genoomsequentie van iedereen
Precision medicine
- Kennis DNA en SNPs => diagnose, preventie…
- Farmacogenomics
= bepaald waarom mensen slecht reageren op drug
- Therapie op maat
- Ethische aspecten
c. 2e generatie: short read NGS
= massief parallel sequencen van geamplificeerde DNA moleculen
+ hoge throughput, lage kost
+ massale sequentiebepaling
+ snel, accuraat
+ weinig input nodig
- nog kortere readlengte
- veel fouten
2
,DOEL: 1 humaan genoom op 1 toestel op 1 dag
3 pijlers
1) Parallele detectie
- Clusters = kopieën van een template
- Klonale in vitro amplificatie van template
- Multiplexing van meerere clusters
2) Miniaturisatie
3) Integratie
= directe detectie
STAP 1: Moet complex zijn
AANMAAK DNA Genoeg versch sequenties hebben
LIBRARY Stel: tekort => bacteriofaag DNA erbij & later weghalen
3
, Types
- WGS
- WES/ taregeted
- Amplicon sequencing
SOMS: tagmentatie & adaptorligatie samen
- Illumina Nextera kit
- Transposase: knippen & ligeren
- PCR cycli => extra barcodes en adaptoren toev
STAP 2: = in parallel verschillende templates kopiëren
KLONALE IN Solid phase bridge amplification
VITRO
AMPLIFICATIE
Emulsie
Rolling circle amplificatie in oplossing
STAP 3: Sequencing by synthesis
SEQUENCING = synthese streng complementair aan template
Nadeel: fouten bij homopolymeren & indels
4
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