GENOME TECHNOLOGY AND APPLICATIONS
INHOUD
1.1 Principles of DNA cloning ........................................................................................................................... 5
1.1.1 In vitro construction of a recombinant DNA molecule .......................................................................... 5
1.1.2 Transformation ...................................................................................................................................... 5
1.1.3 Selective propagation of clones ............................................................................................................. 6
1.1.4 Isolation of recombinant DNA clones .................................................................................................... 6
1.2 Restriction endonucleases ......................................................................................................................... 6
1.2.1 Cleavage................................................................................................................................................. 7
1.3 DNA ligase .................................................................................................................................................. 8
1.4 characteristics of the vector ...................................................................................................................... 8
1.4.1 Origin of replication (ORI) ...................................................................................................................... 8
1.4.2 Most used vectors ................................................................................................................................. 9
1.4.2.1 Plasmids ........................................................................................................................................ 9
1.4.2.2 Bacteriophages ............................................................................................................................. 9
1.4.3 Avoiding recircularisation ...................................................................................................................... 9
1.4.3.1 Use two different restriction enzymes ......................................................................................... 9
1.4.3.2 Dephosphorylate vector ............................................................................................................. 10
1.4.4 Recombinant screening ....................................................................................................................... 10
1.4.4.1 -galactosidase gene complementation ..................................................................................... 10
1.4.4.2 Suppressor tRNA genes ............................................................................................................... 11
1.4.5 Transformation .................................................................................................................................... 11
1.4.5.1 Classical plasmid preparation in E. coli ....................................................................................... 12
1.4.5.2 In vitro packaging ........................................................................................................................ 13
1.4.6 Lambda cloning vectors ....................................................................................................................... 13
1.4.6.1 Cosmid vectors ............................................................................................................................ 14
1.4.6.2 Bacteriophage P1 vectors ........................................................................................................... 14
1.4.6.3 BAC and PAC vectors ................................................................................................................... 14
1.4.6.4 Cloning in YACs (yeast artificial chromosomes) .......................................................................... 15
1.4.6.5 Overview vectors ........................................................................................................................ 15
1.4.6.6 M13 phages ................................................................................................................................ 16
1.4.6.7 Phagemid (phage-plasmid) vectors ............................................................................................ 16
1.4.6.8 Classical site-directed mutagenesis ............................................................................................ 17
2.1 Expression cloning in bacteria.................................................................................................................. 18
2.1.1 Fusion proteins .................................................................................................................................... 19
1
,2.2 Cloning in eukaryotes............................................................................................................................... 19
2.2.1 Double cassette vector and bicistronic vectors ................................................................................... 19
2.2.2 Two types of expression in eukaryotic cells ........................................................................................ 20
2.2.2.1 Transient expression ................................................................................................................... 20
2.2.2.2 Stable expression ........................................................................................................................ 20
2.2.2.3 Semi stable expression cloning using SV40................................................................................. 20
2.3 Expression in insect cells .......................................................................................................................... 21
2.4 Expression cloning using viral vectors ...................................................................................................... 21
2.4.1 Example of a viral vector: γ-Retrovirus ................................................................................................ 21
2.5 Stable expression in mammalian cells ..................................................................................................... 21
2.5.1 Functional complementation .............................................................................................................. 22
2.5.2 Dominant selectable marker ............................................................................................................... 22
2.6 Example of a classical expression cloning vector ..................................................................................... 22
2.7 Self study: new cloning methods ............................................................................................................. 23
2.7.1 Gateway cloning .................................................................................................................................. 23
2.7.1.1 Restriction enzymes vs. gateway ................................................................................................ 23
2.7.1.2 Target sequences for site-specific recombination ...................................................................... 23
2.7.1.3 Site-specific recombinase mechanism ........................................................................................ 24
2.7.1.4 Advantages ................................................................................................................................. 24
2.7.2 Gibson assembly .................................................................................................................................. 25
3.1 Introduction ............................................................................................................................................. 26
3.2 Start material PCR .................................................................................................................................... 27
3.2.1 Genomic DNA ...................................................................................................................................... 27
3.2.2 cDNA: RT PCR ....................................................................................................................................... 27
3.2.3 Primer design ....................................................................................................................................... 27
3.2.4 Temperature cycles ............................................................................................................................. 28
3.2.5 PCR mistakes........................................................................................................................................ 28
3.3 Types of PCR............................................................................................................................................. 28
3.3.1 Nested Primers .................................................................................................................................... 28
3.3.2 Hot start PCR ....................................................................................................................................... 29
3.3.3 Touch down PCR .................................................................................................................................. 29
3.3.4 Inverse PCR .......................................................................................................................................... 29
3.4 (Dis)advantages of PCR ............................................................................................................................ 30
3.4.1 Most important disadvantages of PCR ................................................................................................ 30
3.4.2 Most important advantages of PCR ..................................................................................................... 30
3.5 Cloning of PCR products ........................................................................................................................... 31
3.6 Allele specific PCR .................................................................................................................................... 31
2
, 3.6.1 ARMS assay .......................................................................................................................................... 31
3.6.2 DOP-PCR .............................................................................................................................................. 32
3.6.3 Alu PCR ................................................................................................................................................ 32
3.6.4 Linker primed PCR amplification ......................................................................................................... 32
3.6.5 Site-directed mutagenesis by PCR ....................................................................................................... 33
3.6.5.1 Add-on mutagenesis ................................................................................................................... 33
3.6.5.2 Mismatch primer mutagenesis ................................................................................................... 33
4.1 Model organisms ..................................................................................................................................... 34
4.1.1 Unicellular organisms .......................................................................................................................... 34
4.1.1.1 Bacteria ....................................................................................................................................... 34
4.1.1.2 Saccharomyces cerevisiae ........................................................................................................... 35
4.1.1.3 Mycoplasma genitalium .............................................................................................................. 35
4.1.2 Invertebrates ....................................................................................................................................... 35
4.1.2.1 Caenorhabditis elegans ............................................................................................................... 35
4.1.2.2 Drosophila Melanogaster............................................................................................................ 36
4.1.3 Vertebrates .......................................................................................................................................... 36
4.1.3.1 Zebrafish (Danio rerio) ................................................................................................................ 36
4.1.3.2 Xenopus ...................................................................................................................................... 37
4.1.3.3 Mammalian models .................................................................................................................... 37
4.2 comparative genomics ............................................................................................................................. 37
4.2.1 Explantations for conserved sequences .............................................................................................. 37
4.2.2 conserved sequences .......................................................................................................................... 38
4.3 Evolution .................................................................................................................................................. 39
4.3.1 Evolution of genomes .......................................................................................................................... 39
4.3.1.1 Duplications ................................................................................................................................ 39
4.3.1.2 Chromosomal rearrangements ................................................................................................... 42
4.3.2 Evolution trees..................................................................................................................................... 42
4.3.2.1 Comparison human-chimpansea genome .................................................................................. 42
4.4 Conclusions .............................................................................................................................................. 43
5.1 Introduction ............................................................................................................................................. 44
5.1.1 Personalized medicine ......................................................................................................................... 44
5.1.2 Pharmacogenetics ............................................................................................................................... 44
5.1.3 Aims of pharmacogenetics .................................................................................................................. 45
5.2 Possible explanations for differences in drug response .......................................................................... 45
5.3 Pharmacogenetic variation in drug metabolism ...................................................................................... 45
5.3.1 Oxidation: Cytochrome P-450 ............................................................................................................. 46
5.3.1.1 Genotype-phenotype relationship of the CYP2D6-polymorphism ............................................. 46
3
, 5.3.1.2 Amplichip CYP450 test ................................................................................................................ 46
5.3.1.3 CYP3A4 polymorphism ................................................................................................................ 46
5.3.1.4 Aldehyde dehydrogenase ........................................................................................................... 47
5.3.2 Acetylation: N-acetylation polymorphism NAT-2 ................................................................................ 47
5.3.3 Methylation: Thiopurine S-Methyltransferase .................................................................................... 47
5.4 Genetic differences in drug target ........................................................................................................... 48
5.4.1 Human growth hormone ..................................................................................................................... 48
5.4.2 β1-adrenerg receptor: sensitivity for β-blocking agents ..................................................................... 48
5.4.3 Warfarin ............................................................................................................................................... 48
5.5 Differentiation between subtypes of a disease ....................................................................................... 49
5.5.1 ERBB2 and herceptin ........................................................................................................................... 49
5.6 Preclinical drug development .................................................................................................................. 49
5.7 Expectations from pharmacogenetics ..................................................................................................... 51
5.7.1 Implementation in clinical practice ..................................................................................................... 51
5.7.1.1 Pilot projects for implementation ............................................................................................... 51
5.7.1.2 Interaction genome-diet ............................................................................................................. 51
6.1 Variation in the human genome .............................................................................................................. 52
6.2 Do we know other types of genomic variation? ...................................................................................... 52
6.2.1 Syndromes ........................................................................................................................................... 52
6.2.2 Fluorescent in situ hybridisation (FISH) ............................................................................................... 52
6.2.3 Principle of Array-CGH ......................................................................................................................... 52
6.3 Copy number variation ............................................................................................................................ 53
6.3.1 CNV is a subtype of Structural Variation ............................................................................................. 53
6.3.2 Mechanisms of rearrangements .......................................................................................................... 54
6.3.3 SNP array ............................................................................................................................................. 54
6.3.4 Examples .............................................................................................................................................. 55
6.3.4.1 The Williams-Beuren syndrome .................................................................................................. 55
6.3.4.2 17q21.31 microdeletion syndrome ............................................................................................. 55
7.1 Introduction ............................................................................................................................................. 56
7.2 Identifying novel, as yet unknown genetic disorders .............................................................................. 56
7.2.1 Trio approach....................................................................................................................................... 56
7.3 Would you not like to screen a large set of patients? ............................................................................. 57
4
INHOUD
1.1 Principles of DNA cloning ........................................................................................................................... 5
1.1.1 In vitro construction of a recombinant DNA molecule .......................................................................... 5
1.1.2 Transformation ...................................................................................................................................... 5
1.1.3 Selective propagation of clones ............................................................................................................. 6
1.1.4 Isolation of recombinant DNA clones .................................................................................................... 6
1.2 Restriction endonucleases ......................................................................................................................... 6
1.2.1 Cleavage................................................................................................................................................. 7
1.3 DNA ligase .................................................................................................................................................. 8
1.4 characteristics of the vector ...................................................................................................................... 8
1.4.1 Origin of replication (ORI) ...................................................................................................................... 8
1.4.2 Most used vectors ................................................................................................................................. 9
1.4.2.1 Plasmids ........................................................................................................................................ 9
1.4.2.2 Bacteriophages ............................................................................................................................. 9
1.4.3 Avoiding recircularisation ...................................................................................................................... 9
1.4.3.1 Use two different restriction enzymes ......................................................................................... 9
1.4.3.2 Dephosphorylate vector ............................................................................................................. 10
1.4.4 Recombinant screening ....................................................................................................................... 10
1.4.4.1 -galactosidase gene complementation ..................................................................................... 10
1.4.4.2 Suppressor tRNA genes ............................................................................................................... 11
1.4.5 Transformation .................................................................................................................................... 11
1.4.5.1 Classical plasmid preparation in E. coli ....................................................................................... 12
1.4.5.2 In vitro packaging ........................................................................................................................ 13
1.4.6 Lambda cloning vectors ....................................................................................................................... 13
1.4.6.1 Cosmid vectors ............................................................................................................................ 14
1.4.6.2 Bacteriophage P1 vectors ........................................................................................................... 14
1.4.6.3 BAC and PAC vectors ................................................................................................................... 14
1.4.6.4 Cloning in YACs (yeast artificial chromosomes) .......................................................................... 15
1.4.6.5 Overview vectors ........................................................................................................................ 15
1.4.6.6 M13 phages ................................................................................................................................ 16
1.4.6.7 Phagemid (phage-plasmid) vectors ............................................................................................ 16
1.4.6.8 Classical site-directed mutagenesis ............................................................................................ 17
2.1 Expression cloning in bacteria.................................................................................................................. 18
2.1.1 Fusion proteins .................................................................................................................................... 19
1
,2.2 Cloning in eukaryotes............................................................................................................................... 19
2.2.1 Double cassette vector and bicistronic vectors ................................................................................... 19
2.2.2 Two types of expression in eukaryotic cells ........................................................................................ 20
2.2.2.1 Transient expression ................................................................................................................... 20
2.2.2.2 Stable expression ........................................................................................................................ 20
2.2.2.3 Semi stable expression cloning using SV40................................................................................. 20
2.3 Expression in insect cells .......................................................................................................................... 21
2.4 Expression cloning using viral vectors ...................................................................................................... 21
2.4.1 Example of a viral vector: γ-Retrovirus ................................................................................................ 21
2.5 Stable expression in mammalian cells ..................................................................................................... 21
2.5.1 Functional complementation .............................................................................................................. 22
2.5.2 Dominant selectable marker ............................................................................................................... 22
2.6 Example of a classical expression cloning vector ..................................................................................... 22
2.7 Self study: new cloning methods ............................................................................................................. 23
2.7.1 Gateway cloning .................................................................................................................................. 23
2.7.1.1 Restriction enzymes vs. gateway ................................................................................................ 23
2.7.1.2 Target sequences for site-specific recombination ...................................................................... 23
2.7.1.3 Site-specific recombinase mechanism ........................................................................................ 24
2.7.1.4 Advantages ................................................................................................................................. 24
2.7.2 Gibson assembly .................................................................................................................................. 25
3.1 Introduction ............................................................................................................................................. 26
3.2 Start material PCR .................................................................................................................................... 27
3.2.1 Genomic DNA ...................................................................................................................................... 27
3.2.2 cDNA: RT PCR ....................................................................................................................................... 27
3.2.3 Primer design ....................................................................................................................................... 27
3.2.4 Temperature cycles ............................................................................................................................. 28
3.2.5 PCR mistakes........................................................................................................................................ 28
3.3 Types of PCR............................................................................................................................................. 28
3.3.1 Nested Primers .................................................................................................................................... 28
3.3.2 Hot start PCR ....................................................................................................................................... 29
3.3.3 Touch down PCR .................................................................................................................................. 29
3.3.4 Inverse PCR .......................................................................................................................................... 29
3.4 (Dis)advantages of PCR ............................................................................................................................ 30
3.4.1 Most important disadvantages of PCR ................................................................................................ 30
3.4.2 Most important advantages of PCR ..................................................................................................... 30
3.5 Cloning of PCR products ........................................................................................................................... 31
3.6 Allele specific PCR .................................................................................................................................... 31
2
, 3.6.1 ARMS assay .......................................................................................................................................... 31
3.6.2 DOP-PCR .............................................................................................................................................. 32
3.6.3 Alu PCR ................................................................................................................................................ 32
3.6.4 Linker primed PCR amplification ......................................................................................................... 32
3.6.5 Site-directed mutagenesis by PCR ....................................................................................................... 33
3.6.5.1 Add-on mutagenesis ................................................................................................................... 33
3.6.5.2 Mismatch primer mutagenesis ................................................................................................... 33
4.1 Model organisms ..................................................................................................................................... 34
4.1.1 Unicellular organisms .......................................................................................................................... 34
4.1.1.1 Bacteria ....................................................................................................................................... 34
4.1.1.2 Saccharomyces cerevisiae ........................................................................................................... 35
4.1.1.3 Mycoplasma genitalium .............................................................................................................. 35
4.1.2 Invertebrates ....................................................................................................................................... 35
4.1.2.1 Caenorhabditis elegans ............................................................................................................... 35
4.1.2.2 Drosophila Melanogaster............................................................................................................ 36
4.1.3 Vertebrates .......................................................................................................................................... 36
4.1.3.1 Zebrafish (Danio rerio) ................................................................................................................ 36
4.1.3.2 Xenopus ...................................................................................................................................... 37
4.1.3.3 Mammalian models .................................................................................................................... 37
4.2 comparative genomics ............................................................................................................................. 37
4.2.1 Explantations for conserved sequences .............................................................................................. 37
4.2.2 conserved sequences .......................................................................................................................... 38
4.3 Evolution .................................................................................................................................................. 39
4.3.1 Evolution of genomes .......................................................................................................................... 39
4.3.1.1 Duplications ................................................................................................................................ 39
4.3.1.2 Chromosomal rearrangements ................................................................................................... 42
4.3.2 Evolution trees..................................................................................................................................... 42
4.3.2.1 Comparison human-chimpansea genome .................................................................................. 42
4.4 Conclusions .............................................................................................................................................. 43
5.1 Introduction ............................................................................................................................................. 44
5.1.1 Personalized medicine ......................................................................................................................... 44
5.1.2 Pharmacogenetics ............................................................................................................................... 44
5.1.3 Aims of pharmacogenetics .................................................................................................................. 45
5.2 Possible explanations for differences in drug response .......................................................................... 45
5.3 Pharmacogenetic variation in drug metabolism ...................................................................................... 45
5.3.1 Oxidation: Cytochrome P-450 ............................................................................................................. 46
5.3.1.1 Genotype-phenotype relationship of the CYP2D6-polymorphism ............................................. 46
3
, 5.3.1.2 Amplichip CYP450 test ................................................................................................................ 46
5.3.1.3 CYP3A4 polymorphism ................................................................................................................ 46
5.3.1.4 Aldehyde dehydrogenase ........................................................................................................... 47
5.3.2 Acetylation: N-acetylation polymorphism NAT-2 ................................................................................ 47
5.3.3 Methylation: Thiopurine S-Methyltransferase .................................................................................... 47
5.4 Genetic differences in drug target ........................................................................................................... 48
5.4.1 Human growth hormone ..................................................................................................................... 48
5.4.2 β1-adrenerg receptor: sensitivity for β-blocking agents ..................................................................... 48
5.4.3 Warfarin ............................................................................................................................................... 48
5.5 Differentiation between subtypes of a disease ....................................................................................... 49
5.5.1 ERBB2 and herceptin ........................................................................................................................... 49
5.6 Preclinical drug development .................................................................................................................. 49
5.7 Expectations from pharmacogenetics ..................................................................................................... 51
5.7.1 Implementation in clinical practice ..................................................................................................... 51
5.7.1.1 Pilot projects for implementation ............................................................................................... 51
5.7.1.2 Interaction genome-diet ............................................................................................................. 51
6.1 Variation in the human genome .............................................................................................................. 52
6.2 Do we know other types of genomic variation? ...................................................................................... 52
6.2.1 Syndromes ........................................................................................................................................... 52
6.2.2 Fluorescent in situ hybridisation (FISH) ............................................................................................... 52
6.2.3 Principle of Array-CGH ......................................................................................................................... 52
6.3 Copy number variation ............................................................................................................................ 53
6.3.1 CNV is a subtype of Structural Variation ............................................................................................. 53
6.3.2 Mechanisms of rearrangements .......................................................................................................... 54
6.3.3 SNP array ............................................................................................................................................. 54
6.3.4 Examples .............................................................................................................................................. 55
6.3.4.1 The Williams-Beuren syndrome .................................................................................................. 55
6.3.4.2 17q21.31 microdeletion syndrome ............................................................................................. 55
7.1 Introduction ............................................................................................................................................. 56
7.2 Identifying novel, as yet unknown genetic disorders .............................................................................. 56
7.2.1 Trio approach....................................................................................................................................... 56
7.3 Would you not like to screen a large set of patients? ............................................................................. 57
4
