Technology development for Oligonucleotide Fingerprinting: applying multiplexed PNA hybridizations and MALDI-TOF mass spectrometry detection

Transcription

1 MAX-PLANCK-INSTITUT FÜR MOLEKULARE GENETIK Technology development for Oligonucleotide Fingerprinting: applying multiplexed PNA hybridizations and MALDI-TOF mass spectrometry detection Inaugural-Dissertation zur Erlangung des akademischen Grades "Doktor der Naturwissenschaften (Dr. rer. nat.)" am Fachbereich Biologie - Chemie - Pharmazie der Freien Universität Berlin vorgelegt von Oliver Bauer aus Berlin, Deutschland Berlin Mai 2003

2 Diese Arbeit wurde am Max-Planck-Institut für Molekulare Genetik, Berlin, Deutschland, in der Zeit von Juni 2000 bis Mai 2003 in der Arbeitsgruppe Oligonukleotid- Fingerprinting der Abteilung von Prof. Dr. Hans Lehrach durchgeführt. Der Verfasser versichert, die vorliegende Arbeit eigenständig durchgeführt und alle verwendeten Hilfsmittel angegeben zu haben. 1. Gutachter: Prof. Dr. Hans Lehrach 2. Gutachter: Prof. Dr. Burghardt Wittig Ort und Tag der Disputation: Berlin,

3 To my dear wife Claudia For all your love, support and belief in me

4 Acknowledgements In the first place I particularly thank Prof. Dr. Hans Lehrach for having given me the opportunity to work on this challenging and exciting project, for his advice and for reviewing this thesis. I also thank Prof. Dr. Burghardt Wittig for reviewing this thesis. I am exceptionally grateful to Drs. Uwe Radelof and Michal Janitz for all their encouragement, advice and inspiring discussions as well as the open and independent working environment I could enjoy. For their manifold support and the successful and stimulating team work I should like to express my deep gratitude to Sabine Thamm, Dr. Anna Guerasimova and Beatrix Röhrdanz. I am indebted to Drs. Sascha Sauer, Johan Gobom, and Klaus-Dieter Klöppel for their contribution to the mass spectrometric part of this project, to Dr. Florian Wagner for providing excellent DNA microarraying resources and valuable help and to Dr. Matthias Steinfath for his invaluable bioinformatics support. I am also grateful to my colleagues of the oligonucleotide fingerprinting group for the warm and friendly working atmosphere and to all others I could not mention in this place for their diverse assistance. My special thanks to my dear family and friends for continuous encouragement and mental support. This work was supported by the Bundesministerium für Bildung und Forschung (BMBF) in the course of the 2 nd phase of the German Human Genome Project (DHGP).

5 Contents 1. Summary 1 2. Zusammenfassung 2 3. Introduction General introduction Sequencing by hybridization Oligonucleotide fingerprinting The principle of oligonucleotide fingerprinting Applications of oligonucleotide fingerprinting Assessment of normalization quality Drawbacks of the current process MALDI-TOF mass spectrometry Peptide nucleic acids as hybridization probes A brief introduction to DNA microarrays Objective of this dissertation Materials Laboratory equipment Chemicals Kits Enzymes Molecular weight markers DNA oligonucleotides PNA oligonucleotides Buffers, solutions, and media Methods Clone selection, experimental handling and storage Genomic DNA clones cdna clones DNA plasmid isolation Polymerase Chain Reaction 31 I

6 6.4 Purification of PCR products Agarose gel electrophoresis Photometric determination of nucleic acid concentration DNA PNA DNA sequence verification Experimental handling of PNA Generation of PNA oligonucleotide sets for OFP Charge-tagging of PNA Hybridization of PNA Tube format Slide format Analysis of pure PNA and PNA hybridizations by MALDI-TOF MS Sample preparation Signal acquisition Data analysis Visualization and quality verification Processing Pearson correlation Fabrication and use of DNA microarrays Spotting and processing Fluorescent DNA hybridization and detection Results Characteristics of PNA hybridizations and MALDI-TOF MS based PNA detection Significance of probe length Influence of PNA modifications on hybridization and MALDI-TOF MS detection Determination of total probe number and creation of PNA sets Impact of different parameters on PNA hybridization Influence of probe and target DNA concentration Influence of additives and temperature Multiplexed OFP analysis of selected clones of known sequence Analysis of genomic DNA clones Analysis of cdna clones 57 II

7 7.4 Evaluation of potential DNA immobilization systems for direct hybridization read-out by MALDI-TOF MS Promising surfaces and attachment chemistries Acrylamide-based immobilization system Streptavidin-based immobilization system Nylon-based immobilization system Dendritic immobilization system Comparison of DNA immobilization systems Discussion Parameters of multiplexed PNA hybridizations and subsequent MALDI-TOF MS detection Multiplexed oligonucleotide fingerprinting pilot study Potential MALDI-TOF MS compatible DNA immobilization systems Outlook Appendix References Index of figures/tables Abbreviations Curriculum Vitae Publications Articles/Book chapters Poster presentations 99 III