SAMENVATTING:
GENTECHNOLOGIE
I0O11a
2025-2026
SCHAKELJAAR: BIOCHEMIE EN BIOTECHNOLOGIE
,Lowie Boels Gentechnologie
1. Isolatie en visualisatie van nucleïnezuren ............................................................................................... 6
1. Inleiding ............................................................................................................................................ 6
2. Isolatie van nucleïnezuren .................................................................................................................. 6
Bereiden van bacterieel DNA ............................................................................................................... 6
Opzuiveren van faag DNA .................................................................................................................... 7
CTAB methode voor plant DNA isolatie ................................................................................................ 7
DNA opzuiveren na isolatie .................................................................................................................. 7
Spectrofotometrische zuiverheidsbepaling .......................................................................................... 8
3. Scheiding en detectie van nucleïnezuren ........................................................................................ 8
Agarose gel electroforese .................................................................................................................... 8
DNA staining ....................................................................................................................................... 9
Gelvrije detectie van nucleïnezuren ..................................................................................................... 9
Pulsed field gel electroforese (PFGE).................................................................................................... 9
Immobilisatie van nucleïnezuren ....................................................................................................... 10
4. Speciale vereisen voor werken met RNA ........................................................................................ 11
RNA extractie met paramagnetische parels ........................................................................................ 11
Selectieve mRNA extractie ................................................................................................................ 11
2. Manipulatie van nucleïnezuren ............................................................................................................. 12
1. Inleiding ....................................................................................................................................... 12
Enzymatisch aramentarium voor NZ .................................................................................................. 12
Natuurlijke bronnen van enzymes ...................................................................................................... 12
2. Restrictie van DNA ....................................................................................................................... 12
DNases & nucleases ......................................................................................................................... 12
Exonucleasen ................................................................................................................................... 13
Geschiedenis van restrictie-endonucleasen ...................................................................................... 14
Types van restrictie endonucleasen ................................................................................................... 14
‘Editing’ cleavage sites ...................................................................................................................... 15
3. Ligatie van DNA molecules ........................................................................................................... 15
DNA ligases & Biologische functie ..................................................................................................... 15
Ligase chain reaction (LCR) ............................................................................................................... 16
RNA ligases ...................................................................................................................................... 16
4. DNA modificerende enzymes........................................................................................................ 16
Fosfatases ........................................................................................................................................ 16
Kinases............................................................................................................................................. 17
Terminal deoxynucleotidyl transferase (TdT) ....................................................................................... 17
Topoisomerases................................................................................................................................ 17
Ribonucleases .................................................................................................................................. 18
1
,Lowie Boels Gentechnologie
Site specifieke recombinaties ............................................................................................................ 18
5. Polymerases ................................................................................................................................ 19
RNA polymerasen & reverse transcriptases ........................................................................................ 19
3. PCR & afgeleide technieken.................................................................................................................. 21
1. De polymerase kettingreactie, principe & achtergrond ................................................................... 21
Primer design eigenschappen............................................................................................................ 21
Polymerases voor PCR ...................................................................................................................... 22
Aandachtspunten bij PCR.................................................................................................................. 23
Basisobjectieven .............................................................................................................................. 23
2. Variaties op PCR........................................................................................................................... 24
Tail PCR ............................................................................................................................................ 24
Nested PCR ...................................................................................................................................... 24
Reverse transcriptase PCR ................................................................................................................ 25
Asymetrische PCR ............................................................................................................................ 25
Inverse PCR ...................................................................................................................................... 25
Y-PCR ............................................................................................................................................... 26
Splicing by overlap extension (SOE) ................................................................................................... 27
3. Semi-quantitatieve, competitieve & real-time PCR ........................................................................ 27
Kwantitatieve real time-PCR .............................................................................................................. 27
4. DNA sequentieanalyse als basistechniek voor kloonanalyse .......................................................... 30
Inleiding ........................................................................................................................................... 30
Sanger sequencing ........................................................................................................................... 30
Pyrosequencing ................................................................................................................................ 31
5. Addendum: nieuwe ontwikkelingen in PCR technologie ................................................................. 32
4. Van plasmiden tot basisvectoren .............................................................................................................. 34
1. Introductie ................................................................................................................................... 34
Voorkomen en eigenschappen .......................................................................................................... 34
Fysische transformaties .................................................................................................................... 34
Conjugatie ........................................................................................................................................ 35
2. Een modulaire opbouw: elementen van plasmide vectoren ........................................................... 36
Origin of replication ~ ori locus .......................................................................................................... 36
Selectiemerkers ................................................................................................................................ 38
Regulerende elementen .................................................................................................................... 42
5. Van faagvectoren tot geavanceerde vectoren ..................................................................................... 43
1. Filamentaire bacteriofaagvectoren ............................................................................................... 43
2. Fasmiden..................................................................................................................................... 43
3. Bacteriofaag lambda afgeleide vectoren ....................................................................................... 45
2
, Lowie Boels Gentechnologie
Faag lambda ..................................................................................................................................... 45
Bacteriofaag labmda vectoren ........................................................................................................... 46
4. Kloneringstrategiën voor grote fragmenten ahv faagafgeleide vectoren ........................................... 47
Cosmiden ......................................................................................................................................... 47
P1 faagvectoren ................................................................................................................................ 49
5. Bacterial artificial chromosomes = BACs....................................................................................... 50
BACs ................................................................................................................................................ 50
6. Samenvattend ............................................................................................................................. 50
6. van genklonering tot DNA banken .......................................................................................................... 51
1. Inleiding ....................................................................................................................................... 51
2. Stappen bij het maken van genomische banken ............................................................................ 52
Bereiding van DNA fragmenten .......................................................................................................... 52
Formule van carbon & clarke ............................................................................................................. 52
Ligatiestrategiën & gastheertransformatie .......................................................................................... 52
3. Genoombanken ........................................................................................................................... 54
4. cDNA klonering en cDNA bibliotheken .......................................................................................... 54
cDNA klonering & cDNA bibliotheken ................................................................................................. 54
Basisstrategie voor cDNA klonering ................................................................................................... 55
Methode van gubbler & hoffman ........................................................................................................ 55
cDNA klonering volgens Okayama & Berg ........................................................................................... 56
Rapid amplification of cDNA end (RAcE) ............................................................................................ 56
Random & directionele klonering van cDNA ....................................................................................... 57
Capture techniek voor full lenght cDNA klonering ............................................................................... 57
5. Screening van DNA banken ........................................................................................................... 58
Inleiding ........................................................................................................................................... 58
Directe selectie en identificatie ......................................................................................................... 58
Gen specifieke selectie ..................................................................................................................... 59
6. Voorbeelden ................................................................................................................................ 60
7. Mutagenese ...................................................................................................................................... 61
1. Inleiding ....................................................................................................................................... 61
2. Random mutagenese ................................................................................................................... 61
Transposon mutagenese ................................................................................................................... 61
dNTP analogen ................................................................................................................................. 62
Error prone PCR ................................................................................................................................ 62
Domain shuffling .............................................................................................................................. 63
3. Gerichte (site directed) mutagenese ............................................................................................. 63
Cassette mutagenese ....................................................................................................................... 63
3
GENTECHNOLOGIE
I0O11a
2025-2026
SCHAKELJAAR: BIOCHEMIE EN BIOTECHNOLOGIE
,Lowie Boels Gentechnologie
1. Isolatie en visualisatie van nucleïnezuren ............................................................................................... 6
1. Inleiding ............................................................................................................................................ 6
2. Isolatie van nucleïnezuren .................................................................................................................. 6
Bereiden van bacterieel DNA ............................................................................................................... 6
Opzuiveren van faag DNA .................................................................................................................... 7
CTAB methode voor plant DNA isolatie ................................................................................................ 7
DNA opzuiveren na isolatie .................................................................................................................. 7
Spectrofotometrische zuiverheidsbepaling .......................................................................................... 8
3. Scheiding en detectie van nucleïnezuren ........................................................................................ 8
Agarose gel electroforese .................................................................................................................... 8
DNA staining ....................................................................................................................................... 9
Gelvrije detectie van nucleïnezuren ..................................................................................................... 9
Pulsed field gel electroforese (PFGE).................................................................................................... 9
Immobilisatie van nucleïnezuren ....................................................................................................... 10
4. Speciale vereisen voor werken met RNA ........................................................................................ 11
RNA extractie met paramagnetische parels ........................................................................................ 11
Selectieve mRNA extractie ................................................................................................................ 11
2. Manipulatie van nucleïnezuren ............................................................................................................. 12
1. Inleiding ....................................................................................................................................... 12
Enzymatisch aramentarium voor NZ .................................................................................................. 12
Natuurlijke bronnen van enzymes ...................................................................................................... 12
2. Restrictie van DNA ....................................................................................................................... 12
DNases & nucleases ......................................................................................................................... 12
Exonucleasen ................................................................................................................................... 13
Geschiedenis van restrictie-endonucleasen ...................................................................................... 14
Types van restrictie endonucleasen ................................................................................................... 14
‘Editing’ cleavage sites ...................................................................................................................... 15
3. Ligatie van DNA molecules ........................................................................................................... 15
DNA ligases & Biologische functie ..................................................................................................... 15
Ligase chain reaction (LCR) ............................................................................................................... 16
RNA ligases ...................................................................................................................................... 16
4. DNA modificerende enzymes........................................................................................................ 16
Fosfatases ........................................................................................................................................ 16
Kinases............................................................................................................................................. 17
Terminal deoxynucleotidyl transferase (TdT) ....................................................................................... 17
Topoisomerases................................................................................................................................ 17
Ribonucleases .................................................................................................................................. 18
1
,Lowie Boels Gentechnologie
Site specifieke recombinaties ............................................................................................................ 18
5. Polymerases ................................................................................................................................ 19
RNA polymerasen & reverse transcriptases ........................................................................................ 19
3. PCR & afgeleide technieken.................................................................................................................. 21
1. De polymerase kettingreactie, principe & achtergrond ................................................................... 21
Primer design eigenschappen............................................................................................................ 21
Polymerases voor PCR ...................................................................................................................... 22
Aandachtspunten bij PCR.................................................................................................................. 23
Basisobjectieven .............................................................................................................................. 23
2. Variaties op PCR........................................................................................................................... 24
Tail PCR ............................................................................................................................................ 24
Nested PCR ...................................................................................................................................... 24
Reverse transcriptase PCR ................................................................................................................ 25
Asymetrische PCR ............................................................................................................................ 25
Inverse PCR ...................................................................................................................................... 25
Y-PCR ............................................................................................................................................... 26
Splicing by overlap extension (SOE) ................................................................................................... 27
3. Semi-quantitatieve, competitieve & real-time PCR ........................................................................ 27
Kwantitatieve real time-PCR .............................................................................................................. 27
4. DNA sequentieanalyse als basistechniek voor kloonanalyse .......................................................... 30
Inleiding ........................................................................................................................................... 30
Sanger sequencing ........................................................................................................................... 30
Pyrosequencing ................................................................................................................................ 31
5. Addendum: nieuwe ontwikkelingen in PCR technologie ................................................................. 32
4. Van plasmiden tot basisvectoren .............................................................................................................. 34
1. Introductie ................................................................................................................................... 34
Voorkomen en eigenschappen .......................................................................................................... 34
Fysische transformaties .................................................................................................................... 34
Conjugatie ........................................................................................................................................ 35
2. Een modulaire opbouw: elementen van plasmide vectoren ........................................................... 36
Origin of replication ~ ori locus .......................................................................................................... 36
Selectiemerkers ................................................................................................................................ 38
Regulerende elementen .................................................................................................................... 42
5. Van faagvectoren tot geavanceerde vectoren ..................................................................................... 43
1. Filamentaire bacteriofaagvectoren ............................................................................................... 43
2. Fasmiden..................................................................................................................................... 43
3. Bacteriofaag lambda afgeleide vectoren ....................................................................................... 45
2
, Lowie Boels Gentechnologie
Faag lambda ..................................................................................................................................... 45
Bacteriofaag labmda vectoren ........................................................................................................... 46
4. Kloneringstrategiën voor grote fragmenten ahv faagafgeleide vectoren ........................................... 47
Cosmiden ......................................................................................................................................... 47
P1 faagvectoren ................................................................................................................................ 49
5. Bacterial artificial chromosomes = BACs....................................................................................... 50
BACs ................................................................................................................................................ 50
6. Samenvattend ............................................................................................................................. 50
6. van genklonering tot DNA banken .......................................................................................................... 51
1. Inleiding ....................................................................................................................................... 51
2. Stappen bij het maken van genomische banken ............................................................................ 52
Bereiding van DNA fragmenten .......................................................................................................... 52
Formule van carbon & clarke ............................................................................................................. 52
Ligatiestrategiën & gastheertransformatie .......................................................................................... 52
3. Genoombanken ........................................................................................................................... 54
4. cDNA klonering en cDNA bibliotheken .......................................................................................... 54
cDNA klonering & cDNA bibliotheken ................................................................................................. 54
Basisstrategie voor cDNA klonering ................................................................................................... 55
Methode van gubbler & hoffman ........................................................................................................ 55
cDNA klonering volgens Okayama & Berg ........................................................................................... 56
Rapid amplification of cDNA end (RAcE) ............................................................................................ 56
Random & directionele klonering van cDNA ....................................................................................... 57
Capture techniek voor full lenght cDNA klonering ............................................................................... 57
5. Screening van DNA banken ........................................................................................................... 58
Inleiding ........................................................................................................................................... 58
Directe selectie en identificatie ......................................................................................................... 58
Gen specifieke selectie ..................................................................................................................... 59
6. Voorbeelden ................................................................................................................................ 60
7. Mutagenese ...................................................................................................................................... 61
1. Inleiding ....................................................................................................................................... 61
2. Random mutagenese ................................................................................................................... 61
Transposon mutagenese ................................................................................................................... 61
dNTP analogen ................................................................................................................................. 62
Error prone PCR ................................................................................................................................ 62
Domain shuffling .............................................................................................................................. 63
3. Gerichte (site directed) mutagenese ............................................................................................. 63
Cassette mutagenese ....................................................................................................................... 63
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