Cartilage hair hypoplasia and the RMRP gene [Elektronische Ressource] / Pia Hermanns

Brendan Lee

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2005
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

‘Cartilage Hair Hypoplasia

and the RMRP gene’







Ph.D. Thesis

to accomplish

Doctorate Degree in Science

at the
Faculty of Biology
Johannes Gutenberg-University of Mainz
in Mainz



Pia Hermanns



Mainz 2005





























In memory of my mother



Table of Contents
Table of contents
Index of Figures: ...............................................................................................................................V Tables:..............................................................................................................................VII
1 Introduction.............................................................................................................................1
1.1 Skeletal development ...................................................................................................................1
1.2 Disorders of the Skeleton ............................................................................................................8
1.3 Cartilage Hair Hypoplasia ..........................................................................................................9
1.4 The RMRP gene.........................................................................................................................11
1.5 Specific Aims ..............................................................................................................................14
Methods..................................................................................................................................15
2.1 DNA isolation methods..............................................................................................................15
2.1.1 Genomic DNA isolation from whole blood (from February 2001 to December 2002) .......15
2.1.2 Genomic DNA isolation from whole blood (from January 2003 to present) .......................15
2.1.3 Isolation of genomic DNA from mouse tails for genotyping...............................................16
2.1.4 Isolation of genomic om yeast cells .........................................................................17
2.1.5 Isolation of genomic DNA from ES cell clones for Mini-Southern analysis .......................17
2.1.6 Isolation of plasmid DNA ....................................................................................................18
2.1.6.1 Isolation of plasmid DNA from bacteria for subcloning...............................................................18
2.1.6.2 Isolation of plasmid DNA from bacteria for sequencing ..............................................................18
2.1.6.3 Isolation of plasmid DNA from bacteria for transfection .............................................................19
2.1.6.4 Isolation of plasmid DNA from yeast cells ...................................................................................19
2.2 Total RNA isolation ...................................................................................................................20
2.2.1 Isolation of total RNA from mammalian cells .....................................................................20
2.2.2 Isolation of tom EDTA whole blood.................................................................20
2.2.3 Isolation of total RNA from Saccharomyces cereviseae ......................................................20
2.3. DNA and RNA standard methods...........................................................................................21
2.3.1 Electrophoresis methods.......................................................................................................21
2.3.1.1 Agarose gel electrophoresis for DNA............................................................................................21
2.3.1.2 Agar electrRNA............................................................................................21
2.3.1.3 PAA gel electrophoresis.................................................................................................................22
2.3.2 Purification of DNA fragments from agarose gels...............................................................22
2.3.2.1 Purification of DNA fragments < 10 kb from agarose gels ..........................................................22
2.3.2.2 Purification of ragments >10 kb from agarose gels ...........................................................23
2.4 PCR techniques..........................................................................................................................23
2.4.1 Standard PCR .......................................................................................................................23
2.4.2 Long template PCR ..............................................................................................................24
2.4.3 Site-directed mutagenesis PCR ............................................................................................24
2.4.4 Reverse-Transcriptase ..........................................................................................................25
2.4.5 Quantitative PCR (Real-Time-PCR) ....................................................................................25
2.4.6 Subcloning of PCR products ................................................................................................26
2.5 DNA sequencing.........................................................................................................................26
2.5.1 Dye terminator reaction........................................................................................................26
2.5.2 Dye primer reaction..............................................................................................................27
2.6 Competent cells ..........................................................................................................................27
2.7 Transformation of DNA into bacteria cells .............................................................................27

I Table of Contents
2.8 Manipulation of yeast cells........................................................................................................28
2.8.1 High-efficiency transformation of Yeast..............................................................................28
2.8.2 Generation of yeast knockout construct ...............................................................................28
2.8.3 Sporulation and dissecting of tetrads....................................................................................29
2.8.4 PI staining of yeast cells for FACS analysis ........................................................................30
2.8.5 Phloxine B staining of yeast cells for FACS analysis ..........................................................30
2.8.6 Replica plating of yeast cells................................................................................................31
2.9 Transfection of adherent cells...................................................................................................31
2.10 Firefly luciferase assay in extracts prepared from transfected cells ...................................32
2.11 X-gal assays ..............................................................................................................................32
2.11.1 X-gal assay from cell extracts prepared from transfected cells..........................................32
2.11.2 X-gal staining of lacZ positive newborn mice....................................................................33
2.12 Labeling of DNA and RNA probes with isotopes..................................................................33
2.12.1 Random primed oligo labeling ...........................................................................................33
2.12.2 Endlabeling of chemically synthesized oligos....................................................................33
352.12.3 Labeling of in situ probes with S .....................................................................................34
2.13 Hybridization techniques ........................................................................................................35
2.13.1 Southern Hybridization ......................................................................................................35
2.13.2 Northern Hybr ......................................................................................................35
2.13.2.1 Northern Hybridization with Agarose gels..................................................................................35
2.13.2.2 Northern Hybridization with Polacrylamide gels........................................................................36
2.13.3 In situ Hybridization...................................................