Apex on DNA microarrays: applications in SNP analysis, mutation detection and DNA resequencing

Report
Summary of lectures
List of participants
Photos

Report ... (draft)

The training course "APEX on DNA Microarrays: Applications in SNP Analysis, Mutation Detection and DNA Resequencing" consisted of two parts. From August 27th until August 31st practical hands-on training covering all aspects of Arrayed Primer Extension (APEX) technology was organised in Tartu University/Estonian Biocentre facilities in Tartu. The practical part was followed by a lecture course in Oetpää, September 1-2, aimed at covering different technologies used in genotyping and mutation detection.

Primer extension is the essential step in most genotyping technologies, although different detection methods, solid phases and labels can be used. The main objective of the practical course was to give the participants an opportunity to apply the primer extension (APEX) technology in different applications including:

• Genotyping by SNP analysis
• Mutation detection using Thalassochip v2, which is a system for analysis of ß-thalassemia-causing mutations in the human ß-globin gene
• Resequencing with p53 gene chip to identify mutations in the human tumor suppressor gene p53

Each day of practical training started with a short introductory lecture giving the theoretical background to the experiments. Also, during the experiments instructors explained the steps of practical work. In addition, each evening discussions of the day were organised, which were, according to the feedback received from the participants, one of the best parts of the whole course bringing their different expertise together. A total of 16 training course participants included experts in fields such as "classical" hybridization-based microarrays, use of arrays for microbial genetics and basics of chip technology, but a few participants were quite new to the microarray field. Therefore, the discussions formed a fruitful basis for exchanging ideas and different opinions.

The course participants were divided into three groups and involved in making their own DNA arrays, including the chemical treatment of slides before oligonucleotide attachment, spotting of oligonucleotides onto slides using the microarrayer and post-processing of the microarrays. Each participant performed his/her own template preparation by PCR and APEX reactions. The wet-lab part was followed by analysis of microarray reaction results using the "Genorama" detector and software, which was developed and commercialised by a local biotech company, "Asper Biotech". The images of all microarray experiments were made available for participants by putting them into a special web site, created for this purpose: http://www.array.ebc.ee/Course.

In summary, very good results from practical experiments were obtained from the SNP genotyping system and Thalassochip V2 ß-thalassemia mutation screening. In the case of the p53 resequencing chip, the results were considered good.

The practical training was followed by a high-level lecture course "Theory and Different Practical Methods of Genotyping" in Otepää, where top scientists in the SNP field from Europe and the US presented their work. A brief description of the topics discussed during the course is given below...

Summary of lectures...

Federico Canzian (IARC, Lyon, France) gave an excellent overview of the basis of genotyping, focusing on the theoretical background of population genetics and new frontiers in the genetics of common diseases in order to find genes with high prevalence alleles conferring a low increase or decrease of risk. He gave also an introduction to technologies used for genotyping, with special attention to primer extension methods and developments in SNP research, using them as markers for whole genome association studies.

Ann-Christine Syvänen (Uppsala University, Sweden) and Tomi Pastinen (Montreal Genome Centre, McGill University, Canada) presented new developments in minisequencing, which is another primer extension method on solid surfaces and was developed by A-C Syvänen and her colleagues a couple of years ago.

Michael Phillips (Orchid Biosciences, Inc, USA) discussed the determination of SNP allele frequencies in three defined populations and CEPH pedigrees using primer extension technolog. SNP-IT on multiple platforms is the key component of the next phase of the Human Genome Project at Orchid. The final goal of these studies will be the formation the basis for the first defined human SNP map, which could provide resources necessary to conduct genome-wide linkage and association studies using SNPs.

Mohammad Sohail (Oxford University, UK) presented data about studies of DNA microarray basics and use of oligonucleotide scanning arrays.

Lynn B. Jorde (University of Utah, Salt Lake City, USA) focused on genetic variation, linkage disequilibrium (LD), and the search for complex disease genes. He presented data about worldwide variation of LD, which is found at greater distances in Asian and European populations than in African populations. LD declines with physical distance between markers, although the pattern of this decline is irregular. He discussed the implications of these findings for gene finding strategies and the pros and cons of using isolated populations in association-based gene mapping studies.

Ivo Glynne Gut (Centre National de Genotypage, France) gave a profound overview of SNP genotyping by Mass Spectrometry including the history, current developments and perspectives of matrix-assisted laser desorption ionisation mass spectrometry (MALDI) used for the analysis of biomolecules.

Jörg Hoheisel (DKFZ, Heidelberg, Germany) presented several examples on the development of technologies for the analysis of large genomic areas to entire genomes with respect to the encoded functions and their regulation. He also described DKFZ's efforts in use of protein- and peptide-microarrays.

Joachim W. Engels (Johann Wolfgang Goethe-Universität, Frankfurt/M, Germany) focused his presentation on fluorescence detection on DNA chips and specifically, on new labeling strategies used to attach dyes at the exocyclic aminogroups of A, C and G nucleotides.

Pui-Yan Kwok (Washington University, St. Louis, MI, USA) discussed the possibilities of fluorescence polarisation (FP) detection in nucleic acid analysis and three SNP genotyping approaches used with FP detection: the incorporation of a specific dye-terminator by DNA polymerase; 5'-nuclease cleavage of allele-specific probes; and invasive cleavage of allele-specific flap probes by cleavase.

Heikki Lehväslaiho (EBI, Hinxton, UK) gave an overview of publicly available programs, websites and databases used to analyse and organise sequence variations. He described the efforts at the EBI to create a framework representing a eukaryotic gene, transcribed and translated in a way that allows tracing the effects of (multiple) sequence change(s) up and down DNA, RNA and amino acid levels. Secondly, he described their efforts to store the information in exchangeable formats and to compute the descriptive attributes and thirdly about variations in the genomic context using the location in genomic clones.

More information on the training course and the full abstracts are available at http://www.biotech.ebc.ee/esf-course/index.html#1

List of participants ...

Some photos from the meeting ...

Group photo of participants and speakers, taken in Otepää (below).

Practical course participants and course instructors, taken in front of the Estonian Biocentre, Tartu (below).