Mutation analysis of tissue sections and single cells using low-volume polymerase chain reaction [Elektronische Ressource] / vorgelegt von Veronika Mayer

Veronika Mayer

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 40 Mo

Extrait

Mutation analysis of tissue sections and single cells
using low-volume polymerase chain reaction








Dissertation
der Fakultät für Geowissenschaften
der Ludwig-Maximilians-Universität München




vorgelegt
von
Veronika Mayer




03. März 2010





































Erstgutachter: Prof. Dr. Wolfgang Heckl
Zweitgutachter: PD Dr. Stefan Thalhammer, Universität Augsburg

Tag der mündlichen Prüfung: 12. Juli 2010




Acknowledgement


First of all I would like to thank Prof. Dr. Wolfgang Heckl for supervising my PhD thesis.
I thank PD Dr. Stefan Thalhammer for the possibility of doing my work in his research
group at the Helmholtz Zentrum München.
Special thanks go to Prof. Dr. med. Dipl. chem. Elke Holinski-Feder and the whole MGZ
team for their extraordinary support and mentoring not only in experimental questions.
Many thanks especially to Dr. Ulrike Schön, Dr. Udo Koehler and Christian Neukäufer
for their encouragement and inspiration during the last three years.
My colleagues Teresa Neumaier, Daniela Woide and Anna-Lena Idzko, I want to thank
very much indeed for their friendship and a really great teamwork.
Thanks a lot to all members of my working group and Anita Pedone at the Helmholtz
Zentrum München. Without their continuous help in technical, administrative and
intellectual issues this thesis had not been possible.
I would like to express my gratitude to Prof. Dr. Sarbia for the possibility of taking
pictures of the stained tissue sections and for answering all my questions related to
colon polyps.
Einen riesigen Dank auch an meine Familie und besonders meinem Mann, die immer
für mich da waren.







Contents 1
Contents


1 Abstract ........................................................................... 4
Zusammenfassung .......................................................... 6


2 Introduction ...................................................................... 8

2.1 Molecular genetic diagnostics ......................................................... 8
2.2 Laser microdissection and SPATS .................................................. 8
2.3 Polymerase chain reaction ............................................................ 10
2.4 Colorectal cancer ........................................................................... 11
2.5 Single cell analysis and preimplantation genetic diagnosis .......... 17


3 Abbreviations ................................................................. 19


4 Material and Methods .................................................... 21

4.1 Patients .......................................................................................... 21
4.1.1 Colon polyps .................................................................................................. 21
4.1.2 Familial adenomatous polyposis coli ............................................................. 21

4.2 Preparations for laser microdissection .......................................... 22
4.2.1 Tissue preparation ......................................................................................... 22
4.2.2 Single cell preparation ................................................................................... 23
Contents 2
4.3 Laser microdissection .................................................................... 23

4.4 Single particle transfer via SPATS ................................................ 24

4.5 Control DNA ................................................................................... 26

4.6 Analysis methods .......................................................................... 27
4.6.1 Polymerase chain reactions........................................................................... 27
4.6.1.1 Low-volume multiplex PCR............................................................................ 27
4.6.1.2 Nested PCR................................................................................................... 30
4.6.2 Gel electrophoresis ........................................................................................ 31
4.6.3 Fragment length analysis .............................................................................. 32
4.6.4 Sequencing analysis ...................................................................................... 32


5 Results .......................................................................... 34

5.1 Characterization of colon polyp tissue .......................................... 34
5.1.1 Analysis of mutations in the genes BRAF and KRAS .................................... 34
5.1.2 Analysis of microsatellite instability ................................................................ 53
5.1.3 Comparison of mutation and microsatellite analysis ...................................... 59

5.2 Mutation analysis of single cells .................................................... 63
5.2.1 Efficiency of low-volume multiplex PCR with fixed single cells ...................... 63
5.2.2 Establishment of a single cell analysis system for patient-specific APC
mutations ....................................................................................................... 64



Contents 3
6 Discussion ..................................................................... 68

6.1 Characterization of colon polyp tissue .......................................... 68

6.2 Mutation analysis of single cells .................................................... 84


7 References .................................................................... 91


8 Appendix........................................................................... i
Publications ...................................................................................................... i
One- and Three-letter code of amino acids and RNA codon table .............. xxiii
Curriculum Vitae .......................................................................................... xxv






















Abstract 4
1 Abstract

In modern molecular genetic diagnostics a trend towards very small amounts of DNA
down to the analysis of single cells can be observed in recent years. Therefore,
techniques for precise isolation and transfer of specific sample material are required
prior to analysis.
In this work sample isolation was achieved using laser microdissection in combination
with a low-pressure single particle adsorbing transfer system. Isolated samples were
transferred horizontally to a planar chemically structured polymerase chain reaction
(PCR) glass slide instead of into reaction tubes as is the case in most other
microdissection techniques. On this glass slide, a low-volume PCR in a total reaction
volume of 1 μl is performed. Reduction of the reaction volume has the potential to
dramatically increase the efficiency and sensitivity of PCR compared to PCR in larger
reaction volumes of up to 50 μl. It is therefore applicable to analyses at the single cell
level.

In the first part of this work, three colon polyps of two patients at risk for colorectal
cancer (CRC) were characterized simultaneously regarding mutations of the two proto-
oncogenes BRAF and KRAS and microsatellite instability (MSI) status. Major aspects in
CRC development are microsatellite instability, CpG island methylator phenotype and
mutations in certain genes. The genes BRAF and KRAS are components of the MAPK
ERK signalling pathway and gain-of-function mutations of either of them leads to the
activation of the pathway and therefore to cell proliferation.
Low-volume multiplex PCR directly from formalin-fixed paraffin-embedded polyp tissue
sections (hyperplastic polyp, sessile serrated and tubular adenoma) was performed. It
was demonstrated that hotspot mutations of BRAF and KRAS occurred simultaneously
in the same sample isolated from one polyp of a patient. Furthermore, mutations in both
genes, besides the hotspots, were detected very often in the same samples. In contrast,
it was shown recently that mutations at the hotspots of BRAF (mutation V600E) and
KRAS (codons 12, 13, 59, 61 and 63) are mutually exclusive in precursor lesions of
sporadic microsatellite stable and MSI CRC. Compared to the CRC classification
suggested by Jass, the results obtained in this work indicate an association with the
serrated pathway model comprising mutations in BRAF and KRAS and MSI or
Abstract 5
microsatellite stability. It was shown that characterization of such colon polyps is
important for a better molecula