Samenvatting Moleculaire Biologie
Inhoudsopgave
Introductiecollege ................................................................................................................................... 3
Introduction ............................................................................................................................................ 9
Module 1 Genome organisation and gene structure .......................................................................... 10
Part 1 Tomato genome.......................................................................................................................... 10
Genome Complexity .......................................................................................................................... 10
Agarose gel electrophoresis .............................................................................................................. 12
Measuring DNA/RNA concentration ................................................................................................. 14
Restriction enzymes .......................................................................................................................... 14
Part 2 Gene structure ............................................................................................................................ 16
Exercise 2.1 Determining the exon/intron distribution in an eukaryotic gene ................................. 16
Exercise 2.2 Find the transcription start and stop signals ................................................................. 18
Exercise 2.3 Open reading frame, 5’-UTR and 3’-UTR ....................................................................... 20
Part 3 Gene detection ........................................................................................................................... 22
Polymerase Chain Reaction (PCR) ..................................................................................................... 22
Experiment 1b ................................................................................................................................... 25
Southern blotting .............................................................................................................................. 25
BLAST ................................................................................................................................................. 28
CSI case .............................................................................................................................................. 30
Report Experiment 1a & 1b ................................................................................................................... 31
Experiment 1a ................................................................................................................................... 31
Experiment 1b ................................................................................................................................... 33
Module 4 ............................................................................................................................................... 36
Part 1 Cloning ........................................................................................................................................ 36
Step 1 Digestion and ligation............................................................................................................. 37
Step 2 Transformation of competent E. coli cells ............................................................................. 38
Step 3 Selection of transformants ..................................................................................................... 39
Exercise 4.1 Creating a physical map of your recombinant plasmid ................................................. 41
Part 2 Applications ................................................................................................................................ 42
Genome library .................................................................................................................................. 42
cDNA library....................................................................................................................................... 42
GFP-fusion ......................................................................................................................................... 43
Reporter construct ............................................................................................................................ 44
Expression vector .............................................................................................................................. 44
Report module 4.................................................................................................................................... 46
1 BIC-10306 Practical Biological Chemistry – Moleculaire Biologie
,Module 2 ............................................................................................................................................... 49
Part 1 Transcription ............................................................................................................................... 49
Alternative splicing ............................................................................................................................ 52
Part 2 RT-PCR ......................................................................................................................................... 54
RT-PCR ............................................................................................................................................... 54
RNA isolation ..................................................................................................................................... 54
qPCR .................................................................................................................................................. 56
Part 3 Sequencing .................................................................................................................................. 57
Report module 2.................................................................................................................................... 59
Module 3 ............................................................................................................................................... 62
Part 1 Western blot ............................................................................................................................... 62
Part 2 Mutations.................................................................................................................................... 65
Exercise 3.1 ........................................................................................................................................ 65
Exercise 3.2 ........................................................................................................................................ 65
Report module 3.................................................................................................................................... 66
2 BIC-10306 Practical Biological Chemistry – Moleculaire Biologie
,Introductiecollege
Symbiose is nauwe samenwerking tussen bacteriën of schimmels en planten (bijv. tussen rhizobium
bacteriën en planten als erwt, soja, klaver of pinda, of met arbusculaire mycorrhiza schimmels).
RUBISCO
Ribulose-1,5-biphosphate carboxylase
CO2 in suikers in fotosynthese (planten &
bacteriën, meest voorkomende eiwit ter
wereld)
Genoom organisatie in eukaryoten
Mensen en apen verschillen maar 1.23% in
de genen.
Tomaten genoom
• 12 chromosomen
• Diploïd: 2 kopieën van elk
chromosoom: 2n = 24
• 900.000.000 bp = 900 x 106 bp =
900 x 103 kb = 900 Mb
Organismes op volgorde van genoom grootte (van klein naar groot)
Klein genoom: minste bp. Groot genoom: meeste bp.
1. Arabidopsis 120,000,000 bp
2. Tomaat 900,000,000 bp
3. Kip 1,000,000,000 bp
4. Mens 3,000,000,000 bp
5. Ui 15,000,000,000 bp
6. Amoebe 670,000,000,000 bp
C-value paradox: genoom grootte correleert niet met de complexiteit van een organisme of het aantal
genen
3 BIC-10306 Practical Biological Chemistry – Moleculaire Biologie
, Genomen van (hogere) eukaryoten bevatten veel niet-coderend DNA
Intronen zijn over het algemeen groter dan exonen.
Exonen worden getransleerd naar mRNA coderen voor eiwitten
Human genome: 3 x 109 bp = 3000 Mb
• 22,000 genen
• 19,000 pseudo-genen
• <3 % van het genoom codeert voor eiwit
Eukaryoot DNA bevat heel veel repetitief DNA
Single copy: uniek, komen maar 1x voor.
Spacer DNA: tussen genen, maar wel uniek.
Repetitief DNA (grootste deel) komen meerdere keren voor.
• Functional sequences: functie bekend. Niet 100% hetzelfde maar lijken veel op elkaar.
o Dispersed: op verschillende genomen.
o Tandem: vlak naast elkaar op een genoom.
• Sequence with no known function: “junk”.
4 BIC-10306 Practical Biological Chemistry – Moleculaire Biologie
Inhoudsopgave
Introductiecollege ................................................................................................................................... 3
Introduction ............................................................................................................................................ 9
Module 1 Genome organisation and gene structure .......................................................................... 10
Part 1 Tomato genome.......................................................................................................................... 10
Genome Complexity .......................................................................................................................... 10
Agarose gel electrophoresis .............................................................................................................. 12
Measuring DNA/RNA concentration ................................................................................................. 14
Restriction enzymes .......................................................................................................................... 14
Part 2 Gene structure ............................................................................................................................ 16
Exercise 2.1 Determining the exon/intron distribution in an eukaryotic gene ................................. 16
Exercise 2.2 Find the transcription start and stop signals ................................................................. 18
Exercise 2.3 Open reading frame, 5’-UTR and 3’-UTR ....................................................................... 20
Part 3 Gene detection ........................................................................................................................... 22
Polymerase Chain Reaction (PCR) ..................................................................................................... 22
Experiment 1b ................................................................................................................................... 25
Southern blotting .............................................................................................................................. 25
BLAST ................................................................................................................................................. 28
CSI case .............................................................................................................................................. 30
Report Experiment 1a & 1b ................................................................................................................... 31
Experiment 1a ................................................................................................................................... 31
Experiment 1b ................................................................................................................................... 33
Module 4 ............................................................................................................................................... 36
Part 1 Cloning ........................................................................................................................................ 36
Step 1 Digestion and ligation............................................................................................................. 37
Step 2 Transformation of competent E. coli cells ............................................................................. 38
Step 3 Selection of transformants ..................................................................................................... 39
Exercise 4.1 Creating a physical map of your recombinant plasmid ................................................. 41
Part 2 Applications ................................................................................................................................ 42
Genome library .................................................................................................................................. 42
cDNA library....................................................................................................................................... 42
GFP-fusion ......................................................................................................................................... 43
Reporter construct ............................................................................................................................ 44
Expression vector .............................................................................................................................. 44
Report module 4.................................................................................................................................... 46
1 BIC-10306 Practical Biological Chemistry – Moleculaire Biologie
,Module 2 ............................................................................................................................................... 49
Part 1 Transcription ............................................................................................................................... 49
Alternative splicing ............................................................................................................................ 52
Part 2 RT-PCR ......................................................................................................................................... 54
RT-PCR ............................................................................................................................................... 54
RNA isolation ..................................................................................................................................... 54
qPCR .................................................................................................................................................. 56
Part 3 Sequencing .................................................................................................................................. 57
Report module 2.................................................................................................................................... 59
Module 3 ............................................................................................................................................... 62
Part 1 Western blot ............................................................................................................................... 62
Part 2 Mutations.................................................................................................................................... 65
Exercise 3.1 ........................................................................................................................................ 65
Exercise 3.2 ........................................................................................................................................ 65
Report module 3.................................................................................................................................... 66
2 BIC-10306 Practical Biological Chemistry – Moleculaire Biologie
,Introductiecollege
Symbiose is nauwe samenwerking tussen bacteriën of schimmels en planten (bijv. tussen rhizobium
bacteriën en planten als erwt, soja, klaver of pinda, of met arbusculaire mycorrhiza schimmels).
RUBISCO
Ribulose-1,5-biphosphate carboxylase
CO2 in suikers in fotosynthese (planten &
bacteriën, meest voorkomende eiwit ter
wereld)
Genoom organisatie in eukaryoten
Mensen en apen verschillen maar 1.23% in
de genen.
Tomaten genoom
• 12 chromosomen
• Diploïd: 2 kopieën van elk
chromosoom: 2n = 24
• 900.000.000 bp = 900 x 106 bp =
900 x 103 kb = 900 Mb
Organismes op volgorde van genoom grootte (van klein naar groot)
Klein genoom: minste bp. Groot genoom: meeste bp.
1. Arabidopsis 120,000,000 bp
2. Tomaat 900,000,000 bp
3. Kip 1,000,000,000 bp
4. Mens 3,000,000,000 bp
5. Ui 15,000,000,000 bp
6. Amoebe 670,000,000,000 bp
C-value paradox: genoom grootte correleert niet met de complexiteit van een organisme of het aantal
genen
3 BIC-10306 Practical Biological Chemistry – Moleculaire Biologie
, Genomen van (hogere) eukaryoten bevatten veel niet-coderend DNA
Intronen zijn over het algemeen groter dan exonen.
Exonen worden getransleerd naar mRNA coderen voor eiwitten
Human genome: 3 x 109 bp = 3000 Mb
• 22,000 genen
• 19,000 pseudo-genen
• <3 % van het genoom codeert voor eiwit
Eukaryoot DNA bevat heel veel repetitief DNA
Single copy: uniek, komen maar 1x voor.
Spacer DNA: tussen genen, maar wel uniek.
Repetitief DNA (grootste deel) komen meerdere keren voor.
• Functional sequences: functie bekend. Niet 100% hetzelfde maar lijken veel op elkaar.
o Dispersed: op verschillende genomen.
o Tandem: vlak naast elkaar op een genoom.
• Sequence with no known function: “junk”.
4 BIC-10306 Practical Biological Chemistry – Moleculaire Biologie
