Research interests

Most aspects of systems oriented approaches to the fundamental nature of life, such as molecular evolution and astrobiology, especially concerning computational, experimental and theoretical methods for studying the information processing capabilities of RNA in ancient and modern organisms.

My M.Sc. project has its focus on the modern RNA world as it appears in eukaryotic cells, more specifically on the type of RNA based information processing events known as alternative splicing (see below).

M.Sc. Project Proposal

Title
A Novel Method of Microarray Capture Probe Design for the Analysis of Alternative Splicing

Abstract
A bioinformatic and experimental approach is applied to the development of a novel method of microarray capture probe design that can discriminate between highly similar alternatively spliced mRNA isoforms in order to validate a current method of capture probe design as well as to broaden the range of detectable alternatively spliced mRNA isoforms to include alternative 5’ end and alternative 3’ end splicing, in the nematode C. elegans.

Introduction
Recently the complete genome sequence of various organisms has become available1. Now methods of large-scale expression analysis of gene expression are being developed2, with significant implications for both biology and medicine3.

Alternative splicing of eucaryotic mRNAs can potentially produce vast numbers of mRNA isoforms from the same gene4, each with different biological properties5. A current estimate suggests that at least 35 % of human genes are alternatively spliced6.

Thus alternative splicing is an important factor in mRNA expression analysis since it is crucial not only to identify which genes that are expressed, but also which alternatively spliced mRNA isoforms of a given gene that are produced.

In a current collaboration between the Department of Evolutionary Biology at the University of Copenhagen and the Danish Biotechnology Company Exiqon A/S methods of expression microarrays7 analysis of alternative splicing in the nematode C. elegans are under development.

Preliminary results indicate that the current method of capture probe design is capable of identifying exon skipping and intron retention events.

Purpose
With the possibility to perform microarray analysis of alternative splicing in C. elegans, the purpose of the project is:

I. To develop a method of capture probe design that can discriminate between highly similar alternatively spliced mRNA isoforms.

II. To apply this method on selected genes of C. elegans.

Hence the project will serve both to validate the current method of capture probe design as well as to broaden the range of detectable alternatively spliced mRNA isoforms, to include alternative 5’ end and alternative 3’ end splicing.

The project is part of the collaboration between the Department of Evolutionary Biology and Exiqon A/S, and will be supervised by Professor Peter Arctander and Dr. Daniel C. Jeffares.

Methods
A bioinformatic and experimental approach is applied in the development of microarray capture probe design.

Capture probe sequences for exon skipping and intron retention of genes that are also covered by the current method will be produced in order to compare and validate both methods.

The precise position of the capture probe with respect to the splice site and the effects of modifying the capture probes with LNA8 on the specificity of the capture probes will be tested in collaboration with Nana Jacobsen from Exiqon A/S.

The capture probe testing will be supplemented with other expression analysis methods such as for example northern blots, and RT-PCR based methods.

In order to produce capture probe sequences for large-scale analysis of alternative splicing the method will be automated.

The data from the microarrays will be analysed using different normalisation protocols9 and clustering methods before final interpretation10.

References
1. The C. elegans sequencing Consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science. 282: 2012-2018.

2. Lander ES (1999) Array of hope. Nature genetics (supplementary edition). 21: 3.

3. Liu HX et al (2001) A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nature Genetics. 27: 55-58.

4. Schmucker D et al (2000) Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell. 101: 671-684.

5. Smith CW & Valcarcel J (2000) Alternative pre-mRNA splicing: the logic of combinatorial control. Trends in Biochemical Science. 25: 381-8.

6. Gravely BR (2001) Alternative splicing: increasing diversity in the proteomic world. Trends in Genetics. 17: 100-107.

7. MGED working group on Microarray Data Annotations (2001). Minimum Information About a Microarray Experiment Version 1.0. [From http://www.mged.org]

8. Skouv J & Jakobsen MH (1999) Locked Nucleic Acid (LNA) – a new class of nucleic acid. Phamaceutical Manufactoring International. April: 127-130.

9. Schuchhardt J et al (2000) Normalization strategies for cDNA microarrays. Nucleic Acids Research. 28: E47.

10. Quakenbush J (2001) Computational Analysis of Microarray data. Nature Reviews Genetics. 2: 418-427.