Development and evaluation of multiplex and high-throughput SNP analysis for the ABCA1 gene [Elektronische Ressource] / vorgelegt von Mario C. O. Probst

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• Publié par: universitat_regensburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 2 Mo

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Development and Evaluation of Multiplex
and High-throughput SNP Analysis for the
ABCA1 gene




Dissertation zur Erlangung des Doktorgrades der
Naturwissenschaften
(Dr. rer. nat.)
an der Fakultät für Chemie und Pharmazie
der Universität Regensburg









vorgelegt von
Mario C.O. Probst
Regensburg im März 2004

Development and Evaluation of Multiplex
and High-throughput SNP Analysis for the
ABCA1 gene



Doctoral Thesis
by
Mario C.O. Probst


This work was performed at the Institute of Clinical Chemistry and Laboratory
Medicine at the University of Regensburg between September 1999 and
December 2003 under the supervision of Prof. Gerd Schmitz.



































Date of colloquium: March 29, 2004

Board of examiners: Chairperson: Prof. Hartmut Krienke
First expert: Prof. Otto S. Wolfbeis
Second expert: Prof. Gerd Schmitz
Third expert: Prof. Manfred Liefländer
I














“The aim of science is not to open the door to infinite wisdom, but to set a limit to
infinite error”.
Berthold Brecht, “The Life of Galileo“


II
Acknowledgements
Above all, I would like to thank Prof. Gerd Schmitz for providing the fascinating
technological facilities at his institute. He also gave me the opportunity and
support to visit various conferences and workshops and to participate in several
interesting research projects.
I gratefully acknowledge the support of the head of my working group, Prof.
Charalampos Aslanidis. He was my teacher in molecular biology and he largely
contributed to the completion of this thesis. I am also grateful for the fruitful
cooperation with the former members of our working group: Harald Thumann,
Hajnalka Andrikovics, Marek Bodzioch and Katarzyna Lapicka.
I also acknowledge the support of Dr. Thomas Langmann and the members of
his working group.
For the excellent technical assistance and the good working conditions in our
laboratory, I would like to thank Heidi Kölbl, Dagmar Richter, Ella Schlangstedt,
Andrea Streinbrunner, Ewa Kowalewski and Sabine Witzmann. Especially
Heidi’s assistance contributed in large parts to this thesis.
I should not forget to thank our IT specialists Josef Mages and Markus Solleder.
Josef’s programming skills were essential for the successful establishment of
high-throughput analysis.
Last but not least I would like to acknowledge the help of our engineers Alfons
Gahr and Ruppert Lintl. No fancy machine works without professional
maintenance.
III
Table of Contents
1 Introduction ...........................................................................................................1
2 Background4
2.1 The ABCA1 gene in HDL metabolism..............................................................4
2.1.1 ABC transporters.........................................................................................4
2.1.2 ABCA1 and Tangier Disease.......................................................................4
2.1.3 ABCA1 plays a key role in cellular cholesterol and phospholipid efflux ......5
2.1.4 ABCA1 defects ............................................................................................6
2.1.5 Regulation of ABCA1 ..................................................................................7
2.1.6 Important domains of ABCA1 and interacting proteins................................8
2.2 Formats for SNP identification and analysis..................................................9
2.2.1 Hybridization techniques .............................................................................9
2.2.2 Enzymatic methods ...................................................................................11
2.3 Instrumentation for SNP analysis..................................................................16
2.3.1 Capillary sequencer16
2.3.2 Light Cycler ...............................................................................................18
2.3.3 TaqMan .....................................................................................................21
2.4 Bead-based multiplex analytical platforms ..................................................25
2.4.1 Synthesis, dying and coating of beads......................................................26
2.4.2 The Luminex Technology ..........................................................................27
2.4.3 Assay formats in nucleic acid testing.........................................................28
3 Materials and methods .......................................................................................32
3.1 Patients ............................................................................................................32
3.2 DNA isolation from EDTA blood ....................................................................34
3.3 Study groups for polymorphism identification ............................................34
3.4 Sequence of ABCA1........................................................................................35
3.5 DNA sequencing of PCR products from ABCA1..........................................35
3.6 Fluorescent fragment analysis of VNTR polymorphisms ...........................36
3.7 Electrophoretic mobility shift assay (gel shift assay) .................................36
3.8 Reporter gene assay with ABCA1 promoter.................................................37
3.9 LightCycler SNP analysis...............................................................................37
3.10 TaqMan SNP analysis .....................................................................................38
3.11 Microspheres...................................................................................................38
3.12 Statistical data analysis..................................................................................38
4 Objective ..............................................................................................................40
5 Results .................................................................................................................40
5.1 Screening for functional sequence variations and mutations in ABCA1 ..40
5.1.1 Sequencing of ABCA1 promoter region and exon 1..................................41
5.1.2 Sequencing of exons 49 and 50 of the ABCA1 gene ................................47
5.1.3 Sequencing of the complete ABCA1 gene in individuals with aberrant
HDL levels .................................................................................................47
5.2 High-throughput genotyping of ABCA1 polymorphisms ............................50
5.2.1 LightCycler SNP analysis ..........................................................................52
5.2.2 TaqMan SNP analysis...............................................................................53
5.2.3 High-throughput workflow55
IV
5.2.4 Results of genotyping................................................................................58
5.3 Development of a bead-based multiplex assay............................................62
5.3.1 Design of primers ......................................................................................62
5.3.2 Multiplex-PCR ...........................................................................................63
5.3.3 Allelic discrimination ..................................................................................64
5.3.4 Genotyping results using the multiplex bead assay ..................................67
6 Discussion ...........................................................................................................70
6.1 Screening for functional sequence variations and mutations in ABCA1 ..70
6.2 High-throughput genotyping of ABCA1 polymorphisms ............................73
6.3 Development of a bead-based multiplex assay............................................74
6.4 Final remark.....................................................................................................75
7 Appendix..............................................................................................................77
8 Abbreviations, acronyms and symbols ............................................................91
9 References...........................................................................................................95
10 Summary............................................................................................................109
11 Publications and Patents..................................................................................112
12 Curriculum Vitae................................................................................................116
13 Eidesstattliche Erklärung .................................................................................117

V 1 Introduction 1
1 Introduction