Genome-wide identification and characterization of well-defined genes involved in glaucoma and pterygium corneae [Elektronische Ressource] / vorgelegt von Gabriela Chavarría Soley

Gchavar

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• Gesundheit

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 16
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Genome-wide identification and characterization of well-defined genes
involved in glaucoma and pterygium corneae




Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur
Erlangung des Doktorgrades












vorgelegt von
Gabriela Chavarría Soley
aus San José














Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Universität
Erlangen-Nürnberg















Tag der mündlichen Prüfung: 27.03.2008

Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. Georg Fey
Zweitberichterstatter: Prof. Dr. Andreas Winterpacht











ii

Gedruckt mit Unterstützung des Deuschen Akademischen Austauschdienstes


































iii



















ivIndex
1 Introduction....................................................................................................................... 1
1.1 Glaucoma, general aspects .................................................................................................. 1
1.1.1 Classification of glaucoma .............................................................................................................. 2
1.2 Genetics of POAG ................................................................................................................3
1.2.1 Inheritance and implicated loci ...................................................................................................... 3
1.2.2 Known glaucoma genes .................................................................................................................. 6
1.2.2.1 Myocilin (MYOC) 6
1.2.2.2 OPTN .................................................................................................................................... 7
1.2.2.3 WDR36.. 8
1.3 Primary congenital glaucoma (PCG) ................................................................................. 8
1.3.1 CYP1B1 gene and protein............................................................................................................... 9
1.4 Pterygium corneae ............................................................................................................. 11
1.4.1 Genetics of Pterygium corneae...................................................................................................... 12
1.4.2 Factors implicated in the pathogenesis of pterygium corneae....................................................... 12
2 Methods............................................................................................................................ 14
2.1 Patients................................................................................................................................ 14
2.2 DNA standard methods ..................................................................................................... 14
2.2.1 DNA isolation ............................................................................................................................... 14
2.2.1.1 Salting out procedure for DNA extraction...................................................................... 14
2.2.1.2 Automated DNA isolation................................................................................................. 15
2.2.2 Agarose gel electrophoresis .......................................................................................................... 15
2.2.3 Gel extraction of PCR products.... 15
2.2.4 Quantification of dsDNA .............................................................................................................. 15
2.3 RNA standard methods 15
2.3.1 RNA isolation................................................................................................................................ 15
2.3.2 Quantification of RNA with absorbance at 260nm ....................................................................... 16
2.3.3 Evaluation of RNA quality............................................................................................................ 16
2.4 PCR (polymerase chain reaction), microsatellite analysis, and sequencing ................. 16
2.4.1 Polymerase chain reaction (PCR).................................................................................................. 16
2.4.2 Microsatellite Analysis.................................................................................................................. 16
2.4.3 Purification of PCR products ........................................................................................................ 17
2.4.3.1 Enzymatic purification of PCR products ........................................................................ 17
2.4.3.2 Purification of PCR-products magnetic beads.............................................................. 17
2.4.4 Sequencing of purified PCR products with the Sanger method .................................................... 17
2.4.5 Purification of sequencing products with magnetic beads ............................................................ 18
2.5 Plasmid procedures............................................................................................................ 18
2.5.1 Site-directed mutagenesis.............................................................................................................. 18
2.5.2 Midi Plasmid-DNA-Preparation.19
2.6 Yeast methods..................................................................................................................... 19
2.6.1 Yeast Stocks.................................................................................................................................. 19
2.6.2 Competent yeast cells.................................................................................................................... 19
2.6.3 Yeast transformation ........................................................................................................... 19
2.6.4 Induction of expression and microsome isolation ......................................................................... 20
2.6.5 Microsome Isolation...................................................................................................................... 20
2.6.6 Determination of enzymatic activity .............................................................................................20
2.7 Standard protein methods................................................................................................. 20
2.7.1 Determination of total protein concentration................................................................................. 20
2.7.2 Western Blot.................................................................................................................................. 20
2.8 Bioinformatics tools ........................................................................................................... 21
2.8.1 PCR primer design ........................................................................................................................ 21
2.8.2 Sequencing analysis 21
v2.8.3 Microsatellite Analysis.................................................................................................................. 21
2.8.4 Genome Browsers ......................................................................................................................... 21
2.8.5 Single nucleotide polymorphisms (SNPs) databases..................................................................... 21
2.8.6 Linkage disequilibrium visualization ............................................................................................22
2.8.7 Haplotype reconstruction .............................................................................................................. 22
2.8.8 Multiple sequence alignment......................................................................................................... 22
2.8.9 Expression data analysis......... 22
2.8.10 Gene Ontology ......................................................................................................................... 22
2.9 Nomenclature ..................................................................................................................... 22
2.10 Reagents and Materials ..................................................................................................... 23
2.10.1 Kits ........................................................................................................................................... 23
2.10.2 Instruments .................................................................................