Summary of Scientific Activities
- DNA-Microarray Technology
- Recent technical developments toward the production of high-quality DNA-microarrays
- Label-free and amplification-free detection of microarray analyses by means of peptide nucleic acids (PNAs)
- MolTools - Advanced molecular tools for array-based analyses of genomes, transcriptomes, proteomes and cells.
- Determination of transcription factor binding specificities; applications for identification of genes predisposing to colorectal cancer
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The division works at the development and immediate application of technologies for the production and processing on a genomic scale of biological information that is required for the identification, description and assessment of cellular functions on a molecular level as well as the understanding of the regulation of these processes. Many projects are being done in national and international collaborations.
One emphasis in our efforts is work on DNA-, protein- and peptide-microarrays. Many chemical and biophysical issues as well as matters of data analysis are being addressed in an attempt to understand the underlying procedural aspects, thereby eventually establishing superior analysis procedures.
Based on the technical advances, the methods are immediately put to the test in relevant, biologically driven studies on various organisms. Concerning the analysis of human material, systems are being developed toward early diagnosis, prognosis and evaluation of the success of disease treatment with an accentuation on cancer.
Beside other applications, analyses are performed on the detection and use of disease-relevant single nucleotide polymorphisms (SNPs) in the area of molecular epidemiology, for example. Genotyping of pathogenic organisms or viruses is also being pursued. The analysis of epigenetic variations is another important element of our work.
We also study the variations in transcript levels of all genes of an organism and their phenotypical consequences and compare this data with the actual protein expression by means of complex antibody microarrays. To this end, new processes are required for the selection of the relevant sensor molecules, such as antibodies of high specificity and affinity.
Large-scale mapping and sequencing is still an area of activity, although of decreasing importance. These projects are mostly performed as preparation for subsequent functional analyses. A new area of activity is the establishment of processes for the analysis of the consequences of DNA-structure to enzymatic activities. This direction of research has perspectives for both analytical processes and towards a basic understanding of interactions of protein and nucleic acids.
Apart from publications in scientific journals, the division filed a large number of patents/patent applications, of which several have been licensed out or are being utilised in ongoing collaborations with commercial partners.
DNA-Microarray Technology
With the deciphering of the basic sequence information on a genomic scale being either finished or in an advanced stage for many organisms, experimental procedures for an elucidation of the cellular effects and functional consequences of the DNA-encoded information have become critical for further analyses. In recent years, DNA-microarray technology has emerged as a prime candidate for the performance of many such assays even on a routine basis. The basic methodological arrangement can be adapted to serve as an analytical tool in a large variety of applications.
Recent technical developments toward the production of high-quality DNA-microarrays
Very early experiment on the production of oligonucleotide microarrays by light-directed in situ synthesis. A pattern resembling letters was repeatedly projected onto a glass surface, triggering oligomer synthesis. Subsequenly a complementary, fluorescently labelled oligonucleotide was hybridised to this chip, producing the pattern shown above.
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Different methods for the creation of DNA-microarrays are being used, but the basic idea on how to perform analyses remains the same: hybridisation of an unknown sample to an ordered array of immobilised DNA sensor molecules of known sequence produces a specific hybridisation pattern which can be analysed or compared to a given standard. The sensor molecules consist either of synthetic oligomer or longer, enzymatically generated DNA, mostly PCR-products made from genomic DNA or cDNA clones. Hybridisation techniques, on their own or in combination with methods such as PCR, open up many avenues of genetic analyses.
Very many aspects that influence the production of DNA-microarrays were investigated. Besides normal synthesis procedures, also the photo-controlled in situ synthesis process has been improved dramatically in terms of yield. In addition, methods for the inversion of the oligonucleotides‘ synthesis direction were developed and the relevant monomers synthesised, permitting on-chip polymerase reactions.
Our recently finished work at technical aspects of DNA-microarrays can be sub-divided into the following areas:
Light-controlled in situ synthesis of complex oligonucleotide microarrays of flexible design
In a cooperation, we are using the hardware of 
for the generation and use of complex oligonucleotide arrays. The system permits the in situ synthesis of microarrays containing up to 64,000 different oligonucleotides. All steps necessary for oligomer in situ synthesis (starting from an empty cartridge), sample hybridisation and detection are carried out within the device and on site. Any combination of oligonucleotide sequences can be generated on the microarray, based on individual data files created or assembled by the user. Therefore, empirical results from earlier hybridisations can immediately be applied to the improvement of the next microarray.
In combination with our developments of high-yielding chemistry and inversion of synthesis direction, the device offers a wide range of applications. We use it for various applications such as transcriptional profiling experiments, (multiplex) genotyping experiments for epigenetic measurements, molecular epidemiological studies and virus identification as well as on-chip enzymatic reactions. In addition, the system is being used for various procedures for the production of molecular tools in the MolTools project well beyond the basic production of mere oligonucleotide microarrays.
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Label-free and amplification-free detection of microarray analyses by means of peptide nucleic acids (PNAs)
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Important aspects in microarray analyses are the sensitivity and selectivity of the binding of the assayed DNA molecules. Since the studied DNA-samples usually require a (PCR) amplification and (fluorescence) labelling prior to analysis, time-consuming and costly preparative steps are required, which also might introduce experimental biases. The structural difference between PNA – used as probe on the array – and a DNA-target permits a direct detection of the nucleic acid by mass spectrometry, a process that is much more sensitive than current detection techniques. Thereby, all the preparative steps could be avoided. Upon hybridisation of a DNA or RNA sample to a PNA-array, the phosphates of the nucleid acids can be utilised as an intrinsic label for detection by secondary ion mass spectrometry (SIMS); PNA molecules are lacking phosphate groups entirely. In collaboration with Heinrich Arlinghaus (University of Münster), we have established the processes for analysing genomic DNA directly without the need for amplification or labelling.
Label-free detection by TOF-SIMS analysis. A primary ion beam hits the surface, from which secondary ions, including phosphate fragments, are released. PNA does not contain phosphates. Therefore, phosphate ionsare visible in the mass spectrum of a PNA-microarray only upon hybridisation of nucleic acids.
SNP-typing unlabelled DNA. A dilution series of two different PNA oligomers was spotted left to right (spot diameter: 300 µm) in columns of eight copies on a silicon wafer with gold-surface. Hybridisation was with an unlabelled DNA that was complementary to only the PNA on the right. Analysis was by TOF-SIMS. The image is a false-colour representation of the signal intensities at a mass of 79 Da.
Apart from technical developments with respect to synthesis and detection, also the hybridisation behaviour of DNA samples to PNA arrays is being investigated for a precise understanding of PNA-DNA interactions on solid support. The techniques have a wide range of potential applications: parallel preparation of very many PNA- (or peptide) oligomers for any subsequent use; sequence optimisation of PNA molecules for antisense strategies; and nearly any array-based methods based on the use of oligomers. For the latter purpose, the technology is being pursued further in a collaboration with three commercial partners.
For the production of PNA-arrays, a technique of synthesising PNA oligomers in relatively small quantities but large numbers was established. PNAs are synthesised by an automated process in filter-bottom microtiter plates. The resulting molecules are released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which have been chemically introduced during synthesis. Thus, only full-length PNA-oligomers are attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, do not bind. Different surface chemistries and fitting modifications of the PNA terminus were established. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Based on the results and experience obtained from the synthesis of PNA oligomers, the in situ synthesis and application of peptide arrays is being worked at.
Related Papers
- Matysiak et al. (2001) BioTechniques 31, 896-904. (PDF | 126,91 KB)
- Brandt et al. (2003) Nucleic Acids Res. 31, e119. (PDF | 285,22 KB)
- Bauer et al. (2003) Comp. Funct. Genom. 4, 520-524. (PDF | 225,56 KB)
- Jacob et al. (2003) Peptide Nucleic Acids (Nielsen, P.E., ed.), 261-279.
- Jacob et al. (2004) Methods in Mol. Biol. (Niemeyer, C., ed.), 283-294.
- Arlinghaus et al. (2004) Appl. Surface Sci. 231-232, 392-396. (PDF | 189,18 KB)
- Brandt & Hoheisel (2004) Trends Biotechnol. 22, 617-622. (PDF | 233,07 KB)
- other publications and patents
MolTools - Advanced molecular tools for array-based analyses of genomes, transcriptomes, proteomes and cells.
MolTool Participants:
The MolTools consortium started on January 2004 as a joint research programme bringing together 12 leading European academic groups, five biotech SMEs and one US laboratory working in the area of postgenomic technology development. The partners have pioneered a series of important molecular techniques and will now work together to establish next-generation tools for molecular analysis. Molecular technologies are in a very rapid state of development, the scope for improvement is extreme, and methods are clearly rate limiting for the progress of biology and biotechnology generally. This project represents an important initiative to integrate leading European scientists active in an area of technology development which is central to modern biology.Scientific aims are the establishment of genome analysis technologies set to monitor extensive molecular repertoires, and with the capacity to investigate even single molecules. To this ends, powerful array-based research tools are developed to examine DNA, RNA and proteins. For a description of the research of the consortium as a whole, please, go to the overall MolTools websitea.
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Overall coordination rests with Ulf Landegren at Uppsala University. Six interrelated analytical nodes or workpackages (WPs) have been established. Each WP involves several of the partners, most of whom again participating in several WPs.
WP1: coordinator Marc Zabeau
WP2: coordinator Ivo Gut
WP3: coordinator Hans Lehrach
WP4: coordinator Jörg Hoheisel
WP5: coordinator Jorn Koch
WP6: coordinator Olli Kallioniemi
We are working on projects as part of WP2 (DNA analysis), WP3 (transcript profiling) and WP4 (protein analysis).
Determination of transcription factor binding specificities; applications for identification of genes predisposing to colorectal cancer
Determination of the sequence of the human genome, and knowledge of the genetic code through which mRNA is translated have allowed rapid progress in identification of mammalian proteins. However, less is known about the molecular mechanisms that control expression of human genes, and about the variations in gene expression that underlie many pathological states, including cancer. This is caused in part by lack of information about the 'second genetic code' - binding specificities of transcription factors.
Deciphering this regulatory code is critical for cancer research, as little is known about the mechanisms by which the known genetic defects induce the transcriptional programs that control cell proliferation, survival and angiogenesis. In addition, changes in binding of transcription factors caused by single nucleotide polymorphisms (SNPs) are likely to be a major factor in many quantitative trait conditions, including familial predisposition to cancer.
We aim to develop novel genomics tools and methods for determination of transcription factor binding specificity. These tools will be used for identification of regulatory SNPs that predispose to colorectal cancer, and for characterization of downstream target genes that are common to multiple oncogenic transcription factors.
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