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Samenvatting Biotechnologie
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BiotechnologieSamenvatting
,Inhoud
1 Inleiding..................................................................................................................................................... 3
2 Genetisch materiaal................................................................................................................................... 3
2.1 Structuur DNA..................................................................................................................................... 3
2.1.1 Elementen van de DNA-molecule................................................................................................. 3
2.1.2 Verschil genetisch materiaal van prokaryoten..............................................................................4
2.2 DNA replicatie..................................................................................................................................... 4
2.3 Eiwitsynthese...................................................................................................................................... 5
2.3.1 DNA-transcriptie........................................................................................................................... 5
2.3.2 Enzymen en nucleïnezuren........................................................................................................... 7
2.3.3 DNA-translatie.............................................................................................................................. 9
2.4 Extrachomasaal DNA......................................................................................................................... 10
2.4.1 Mitochondriaal en (chloro)plasten DNA.....................................................................................10
2.4.2 Plasmiden................................................................................................................................... 10
2.5 Expressie van genen.......................................................................................................................... 10
2.5.1 Chromosoomniveau................................................................................................................... 10
2.5.2 Transcriptie niveau..................................................................................................................... 11
2.5.3 RNA-processing niveau............................................................................................................... 11
2.5.4 Translatie niveau........................................................................................................................ 11
2.5.5 Proteïneprocessing en degradatie niveau..................................................................................11
3 Veranderingen in genetisch materiaal..................................................................................................... 11
3.1 Veranderingen in de genen............................................................................................................... 12
3.2 Veranderingen in chromosomen....................................................................................................... 13
4 Biotechnologische technieken................................................................................................................. 13
4.1 PCR (polymerase chain reaction)....................................................................................................... 13
4.1.1 Principe...................................................................................................................................... 13
4.1.2 Evaluatie van de PCR producten................................................................................................. 14
4.1.3 Amplificeren van RNA (RT-PCR).................................................................................................. 15
4.1.4 Kwantitatieve PCR (qPCR)........................................................................................................... 15
4.1.5 Andere PCR technieken.............................................................................................................. 16
4.2 DNA sequentie analyse..................................................................................................................... 17
4.2.1 Eerste generatie sequentie-analyse............................................................................................ 17
4.2.2 Tweede generatie sequencing.................................................................................................... 17
4.2.3 Derde generatie sequencing....................................................................................................... 18
1
,4.3 Genetisch profiel, DNA fingerprinting............................................................................................... 19
4.3.1 RFLP fingerprinting..................................................................................................................... 19
4.3.2 RAPD.......................................................................................................................................... 19
4.3.3 AFLP........................................................................................................................................... 19
4.4 Eiwit onderzoek................................................................................................................................. 20
4.4.1 Samenstellende aminozuren...................................................................................................... 20
4.4.2 Immunoblotting – Western blotting...........................................................................................20
4.4.3 ELISA.......................................................................................................................................... 21
4.4.4 Snelle antigen test (RAT)............................................................................................................ 22
4.5 Recombinant DNA technologie......................................................................................................... 22
4.5.1 RNA interferentie (iRNA of RNAi)............................................................................................... 23
4.5.2 CRISPR/Cas................................................................................................................................. 23
2
,1 Inleiding
Biotechnologie gebaseerd op biologie
Klassieke biotechnologie
o Traditionele technieken
o Kweken van plant en dier, gebruik van bacteriën, gisten en schimmels
Moderne biotechnologie
o Eigenschappen van bacteriën, planten en dieren aanpassen door ingrijpen in DNA
Toepassingen
o Geneeskunde = rode biotechnologie
Vaccins
Geneesmiddelen
Insuline via GGO’s
o Landbouw = groene biotechnologie
Bt-maïs
Opbrengsten verhogen
Schade door insecten en knaagdieren verminderen
o Voeding
Brood bakken
Kaasproductie enzym chymosine
o Industrie = witte biotechnologie
Waspoeders
Papier bleken
Leerlooien
o Wetenschappelijk onderzoek
Inzicht verwerven hoe zit het leven in elkaar
2 Genetisch materiaal
In elke cel 46 chromosomen dragers van DNA
2.1Structuur DNA
2.1.1 Elementen van de DNA-
molecule
DNA
o Desoxyribonucleïnezuur
o Structuur dubbele helix
Aaneenschakeling van nucleotiden
Desoxyribose
Fosfaatgroep
Purine of pyrimidine base
o Pentosesuiker 5 C-atomen
Op 2’ een H
3
, Op 5’een koolstofsuiker
Op 1’ purine of pyrimidine base
o Complementaire basen waterstofbruggen
Grote purine-basen
Adenine (A)
Guanine (G)
Kleine pyrimidine-basen
Cytosine (C)
Thyamine (G)
Opbouw van een DNA molecule
o Toevoegen van desoxyribonucleosidetrifosfaat
Gebonden aan 5’
Splitst 2 fosfaatgroepen af
Hecht aan 3’
o Aangroei telkens in de 5’-3’ richting
2.1.2 Verschil genetisch materiaal van prokaryoten
Prokaryoot Eukaryoot
- DNA in cytoplasma - DNA in kern
- Ringvormig - Dubbele helix
- Geen 5’ of 3’ uiteinde - 5’ en 3’ uiteinde
- Replicatie in cytoplasma - Replicatie in de nucleus
DNA heeft 2 functies
o (Zelf)replicatie = behoud van info
o Eiwitsynthese (transcriptie) = aflezen info voor gebruik
2.2DNA replicatie
Replicatie = verdubbeling van het DNA in de cel
o Om te verdelen over de dochtercellen
o Uit 1 molecule ontstaan 2 identieke moleculen
2 fasen
o Voorbereiding
o Effectieve replicatie
DNA replicatie
1. Replicatiestartpunt = origin/ori initiator proteïnen binden zich hier en maken dit punt herkenbaar
voor andere enzymen
2. Verbreken van de dubbele helix = denaturatie enzym helicase vormt de replicatiebel = 2 vorken
3. Stabilisatie door enkelstrengsbindingsproteïnen
4. Topoïsomerase zorgt voor relaxtie DNA opnieuw dubbele helix
5. Eigenlijke replicatie: DNA polymerase III zorgt voor invoeging van nucleotiden en proofreading
6. Aanvang vereist reeds een P-suikergroep om te binden aan 3’
7. Oplossing ander enzynm, DNA primase, maakt stukje RNA (= primer)
8. DNA-polymerase kan starten met verlengen van de primer
Leading streng en lagging streng
Leading streng Lagging streng
- Primer door DNA primase = start - Primer door DNA primase = start
- DNA-polymerase III 5’-3’ (continu) - DNA polymerase III 5’-3’ (discontinu -
- DNA-polymerase I: RNA-primer DNA Okazakifragmenten) 4
- 2 strengen aan elkaar door ligase - DNA-polymerase I: RNA-primer
exonuclease DNA
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