Dissertation zur Erlangung des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät III

Chemie, Pharmazie und Werkstoffwissenschaften der Universität
des Saarlandes

Schlagworte zum Inhalt (Deutsch):
DNA Methylierung
Molekulare Haplotypisierung
Massenspektrometrie
Pyrosequenzierung
Hochdurchsatz

Zusammenfassung
Methoden, die genetische und epigenetische Variationen analysieren, werden helfen die komplexen Zusammenhänge zwischen dem von der DNA beschriebenen genetischen Code, Genfunktion und -regulation zu entschlüsseln. Dies wird dazu beitragen, die molekulare Grundlage einer Vielzahl von Krankheiten zu identifizieren. In dieser Arbeit wurde das erste Verfahren zur molekularen Bestimmung von Haplotypen (Phase der Allele von Single Nucleotide Polymorphisms) entwickelt, das sich für automatisierte Hochdurchsatzverfahren eignet. Ein neues DNA Polymerasensystem erlaubt die routinemäßige allel-spezifische Amplifikation in bisher nicht gekannter Einfachheit und Länge. Die so hergestellten haploiden Fragmente können mittels Sanger-Sequenzierung oder in einem auf MALDI Massenspektrometrie als Detektionsmethode basierendem Assay nachgewiesen werden. Zwei neue Methoden zur Analyse und präzisen Quantifizierung von DNA Methylierung in mit Natriumbisulfit behandelter DNA wurden entwickelt. Der GOOD Assay zur Epigenotypisierung beruht auf einer quantitativen Abfrage der Allele mittels Primerextension und MALDI massenspektrometrischer Detektion. Für die Analyse von Methylierungspositionen in unmittelbarer Nähe wurde eine komplementäre Methode basierend auf der Pyrosequenzierungstechnologie entwickelt. Für beide Verfahren wurde die Notwendigkeit nachgewiesen, Kalibrierungskurven aufzunehmen, um für eine eventuelle präferenzielle Amplifikation in der PCR zu korrigieren. Bisulfit behandelte DNA, die aus einer Biopsieprobe isoliert wurde, konnte mit hoher Präzision mit beiden Methoden analysiert werden. Der GOOD Assay wurde darüber hinaus im Hochdurchsatz zur Kartierung von Positionen im MHC verwendet, die einen unterschiedlichen Grad an DNA Methylierung in verschieden Geweben aufwiesen.

Schlagworte zum Inhalt (Englisch)
DNA methylation
haplotyping
mass spectrometry
pyrosequencing
high throughput


Abstract
Tools for the analysis of genetic and epigenetic variations will help decipher the complex interrelation between the genetic code, gene function, and regulation. This will support the identification of the genetic basis of disease-associated phenotypes. The first high-throughput method for the direct physical determination of haplotypes, the phase of alleles of individual polymorphisms, in genomic regions of substantial size was developed. It is based on a novel highly allele-specific DNA polymerase system and subsequent analysis by either conventional sequencing or detection of primer extension products by MALDI mass spectrometry. Two novel assays for the accurate analysis of DNA methylation variable positions (MVPs) and their precise quantification on bisulphite treated genomic DNA are presented. The GOOD assay for epigenotyping is based on primer extension for allele-discrimination and MALDI mass spectrometric detection. The multichannel analysis capabilities allow detection of reaction products created in simplex and multiplex reactions. The Pyrosequencing technology was evaluated as a complementary tool for analysis and quantitation of closely neighbouring MVPs. For both methods calibration curves are necessary to account for possible biases during PCR, respectively PCR and primer extension reaction. Bisulphite treated DNA from cancerous tissue biopsies was analysed with high accuracy and resolution using either method. The mass spectrometry based assay was applied in a high-throughput format to the first generation of a draft epigenome map in different tissue types at loci within the major histocompatibility complex.

Analysis of allele-specific amplified PCR products by sequencing and mass spectrometry
Sequencing and mass spectrometry were used for the analysis of haplotypes in this thesis, but products generated by the AccuPrimeTM Taq or similar polymerase systems will have great influence on the analysis of haplotypes by a broad variety of detection platforms. Sequencing of allele-specific amplificates generated sequencing data of the same quality as regular sequencing of diploid fragments. Thus, this approach constitutes thus a new and easy way to determine physical haplotypes by sequencing without the need for the laborious procedures of cloning. This method has wide applicability due to the limited utility of database SNPs, which often fail to define and distinguish between haplotypes and only a fraction of the variants found by resequencing the genomic region of interest are found in databases. Genotypes and haplotypes of the genomic region can be unambiguously identified and/or verified in the studied population by resequencing without the requirement to study extensive pedigrees even in cases where statistical and computational methods fall short.
Combining allele-specific PCR with SNP genotyping and MALDI mass spectrometric detection is an ideal match and opens the door for molecular haplotyping at high throughput in the absence of family information. The major strength of MALDI mass spectrometry is the speed and accuracy of analysis and multiplex potential.


Epigenotyping at high throughput in the major histocompatibility complex

The GOOD assay for epigenotyping was successfully miniaturised and integrated with automated liquid handling and data accumulation. […] PCR replicates were only used for this first proof of principle and reproducibility, but as demonstrated in table 3.11 do not increase accuracy significantly. A triplex primer extension format was chosen for the analysis of MVPs in the amplificates. […] Even in the case of sequence identity masses of the products can be unambiguously assigned in a triplex format by applying different charge tags (3.1). Automated assay design, standard assay conditions and automated data collection and interpretation are critical for any high-throughput method. All assays are carried out under uniform conditions facilitating parallel processing of different amplificates. […]. The developed method is a fast, reliable and high-throughput assay for the accurate analysis and quantification of DNA methylation. In the framework of the EU project “Pilot study for a human epigenome project” this assay will be applied to 80 other PCR products from loci of the MHC.