Summary
Summary Genome Technology and Applications
- Course
- Institution
This is a summary of the teachings of Prof Guy Van Camp, Frank Kooy and Wim Van Hul, based on the slides and notes.
[Show more]
Seller
Follow
BMWstudent19
Reviews received
Content preview
GENOME TECHNOLOGY AND APPLICATIONSINHOUD
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
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller BMWstudent19. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $9.77. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
64438 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 14 years now