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Samenvatting biotechnologie
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BIOTECHNOLOGIE2023-2024
FARMACEUTISCH EN BIOLOGISCHE LABORATORIUM TECHNOLOGIE
,Lowie Boels Biotechnologie
1. Recombinant technologie en vector systeem .......................................................................7
1.1. Basisprincipe van de recombinant DNA technologie .................................................7
1.2. Biologische gastheren in moleculaire biotechnologie ................................................7
1.3. Ontwikkeling van een vector .....................................................................................7
1.4. Enzymen betrokken bij klonering...............................................................................8
1. Restrictie enzymen (RE)............................................................................................8
2. Ligatie van vector met insert DNA ........................................................................... 10
3. Fosfatase enzymen ................................................................................................ 10
4. DNA ligasen ........................................................................................................... 11
1.5. DNA inbreng methode voor gastheercellen ............................................................. 11
1. Chemische inbrengmethode: CaCl2-hitte transformatie ......................................... 11
2. Fysische inbrengmethode: DNA elektroporatie ........................................................ 12
3. Opgroeien/herstel van de getransformeerde cellen ................................................. 12
1.6. Kolonie screening en selectie merkers .................................................................... 13
1. Werking van transpeptidase ................................................................................... 13
2. Werking van ampicilline ......................................................................................... 13
3. Werking van tetracycline ........................................................................................ 14
4. Werking chlorampfenicol ....................................................................................... 14
5. Gebruik van auxotrofe selectie ............................................................................... 15
1.7. Courante kloneringsvectoren .................................................................................. 15
1. pBR322.................................................................................................................. 15
2. pUC18/19 kloneringsvector .................................................................................... 16
2. Nucleïnezuur scheidingstechnieken ........................................................................... 19
2.1 Algemene principes nucleïnezuur opzuiveringstechnieken ...................................... 19
2.2 Chromosomaal DNA isolatie uit prokaryoten ........................................................... 19
1. Pelletisering ........................................................................................................... 19
2. Het ruwe celextract ................................................................................................ 20
2.3 Opzuivering van DNA uit het ruwe extract ................................................................ 22
1. Overzicht van de 3 methode voor de opzuivering ..................................................... 22
2. uitzouten van eiwitten ............................................................................................ 22
3. Fenyl-chloroform-isoamylalcohol extractie = organische extractie ........................... 22
4. Silica gel gebaseerde spin kolommen = solid phase extraction ................................. 24
2.4 Plasmide DNA isolatie uit prokaryoten .................................................................... 24
2.5 Eukaryote genomische DNA-isolatie ....................................................................... 25
1. Extractie uit bloedstalen ......................................................................................... 26
2. Extractie uit weefsel ............................................................................................... 26
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,Lowie Boels Biotechnologie
2.6 Nucleinezuur opzuivering door kolomchromatografie .............................................. 27
1. Anion uitwisselingschromatografie ......................................................................... 27
2. Grootte exclusie chromatografie ............................................................................. 28
3. Solid phase reverse immobilisation beads .............................................................. 28
2.7 RNA opzuiveringstechnieken .................................................................................. 29
2.8 Kwantificatie van DNA en RNA ................................................................................ 30
1. Kwantitatieve bepaling door UV absorptie ............................................................... 30
2. Nanodrop .............................................................................................................. 31
3. Qubit fluorometer .................................................................................................. 32
2.9 Fysiochemische eigenschappen van DNA ............................................................... 33
1. Denaturatieproces van een DNA duplex .................................................................. 33
2. Smeltgedrag en smeltcurve .................................................................................... 33
2.10 Gel elektroforese van nucleïne zuren ...................................................................... 35
1. Soorten gels........................................................................................................... 35
2. Loading dye ........................................................................................................... 36
3. Visualisatie van DNA en RNA moleculen ................................................................. 37
4. Fotografie en gel scanners ...................................................................................... 37
2.11 Kwantificatie DNA en RNA ...................................................................................... 38
1. Conformatie van plasmide DNA in gel-elektroforese ................................................ 39
2. RNA gel elektroforese ............................................................................................. 39
2.12 Capillaire gel elektroforese ..................................................................................... 40
1. Opstelling .............................................................................................................. 40
2. Werking ................................................................................................................. 41
3. Directe detectie door UV absorptie ......................................................................... 41
4. Detectie door LIF ................................................................................................... 41
5. CGE analyse van DNA ............................................................................................ 42
6. Microcapillaire elektroforese (MCE) ........................................................................ 42
2.13 Recuperatie van nucleïnezuren uit de gel ................................................................ 42
3. Polymerase chain reaction (PCR) ................................................................................ 43
3.1. essentiële componenten en PCR-reactiestappen .................................................... 43
3.2. Kritische factoren bij het uitvoeren van PCR ............................................................ 44
1. Controles .............................................................................................................. 44
2. Oorzaken van contaminatie en preventiemaatregelen ............................................. 44
3.3. Cycli aantal – plateau effect ................................................................................... 44
3.4. Thermostabiele DNA polymerasen.......................................................................... 44
3.5. De primerparen...................................................................................................... 45
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, Lowie Boels Biotechnologie
1. Keuze van de primers ............................................................................................. 45
2. 5’ adaptor primers .................................................................................................. 46
3. De annealingstemperatuur Ta van een primer .......................................................... 46
3.6. Het reactiemidden – de Mg2+ concentratie ............................................................. 47
3.7. De matrijs .............................................................................................................. 47
3.8. Restriction fragment lenght polymorphism & PCR (RFLP – PCR) .............................. 48
3.9. Multiplex PCR ........................................................................................................ 48
1. In detectie van pathogene infectie .......................................................................... 48
2. Multiplex PCR in detectie vajn populatie neushoorns in azie en afrika ...................... 48
3. Multiplex PCR in microbioom analyse ..................................................................... 49
3.10. Aspecifieke amplicons ....................................................................................... 50
3.11. Verhoging van de PCR specificiteit ...................................................................... 50
1. Cold start PCR ....................................................................................................... 50
2. Hot start PCR ......................................................................................................... 50
4. Nested PCR ........................................................................................................... 51
5. Touchdown PCR ..................................................................................................... 52
3.12. Kolonie PCR ....................................................................................................... 52
4. Enzymen van de recombinant DNA technologie .......................................................... 54
4.1. Nucleasen ............................................................................................................. 54
1. Algemeen .............................................................................................................. 54
2. Enzymatische fragmentatie voor DNA ..................................................................... 54
3. Enzymatische fragmentatie voor RNA ..................................................................... 55
4. Reverse transcriptase............................................................................................. 55
5. Terminaal deoxynucleotidyl transferase (TdT) .......................................................... 56
4.2. Het immuunsysteem van bacteriën ........................................................................ 57
1. Restrictie modificatie systemen .............................................................................. 57
2. CRISPR CAS systeem in bacteriën en archea........................................................... 57
4.3. Restrictie-endonucleasen ...................................................................................... 58
1. Nomenclatuur ....................................................................................................... 58
2. Type restrictie enzymen .......................................................................................... 58
3. Karakteristieken van type II endonucleasen ............................................................. 58
4. perfecte palindromen............................................................................................. 59
5. Aanmaak van recombinante constructen ................................................................ 60
4.4. Restrictie analyse en fysische kartering................................................................... 60
4.5. Bijzondere aspecten............................................................................................... 62
1. Isocaudomeren, isoschizomeren en neoschizomeren ............................................. 62
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