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

Report
Summary of lectures
List of participants
Photos

Report

Primer extension is the essential step in most genotyping technologies, although different detection methods, solid phase 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, which is a system for analyzing
  ß-thalassemia causing mutations in the human ß-globin gene
• Resequencing with p53 gene chip to identify mutations in the human tumor suppressor gene TP53.

Each day of the practical training started with a short introductory lecture providing theoretical background to the experiments. Also, during the experiments instructors explained in detail the steps of practical work. In addition, discussions of the day were organized every evening, which were according to the feedback received from the participants one of the best parts of the whole course enabling to bring their different expertise together.

The participants of the practical training were divided into three groups based on their wish to perform one out of three APEX technology applications and were involved in making their own DNA arrays, including the silanization of slides before oligonucleotide attachment, spotting of oligonucleotides onto slides using microarrayer and post-processing of microarrays including DNA array quality control. Each participant performed his/her own template preparation by PCR and APEX reactions. The wet-lab part was followed by the analysis of microarray reaction results using "Genorama QuattroimagerÔ" detector and "GenoramaÔ 4.0 software, that has been worked out and commercialized by a local biotech company Asper Biotech Tartu, Estonia). The images of all microarray experiments were made available for participants by writing them all into CD-s together with
"GenoramaÔ4.0" software and limited user key and were sent to the participants shortly after the course.

A practical demonstration of the PyrosequencingÔ instrument and the use of the technology were also organized within the practical training course.

Summary of lectures...

Federico Canzian (IARC, Lyon, France) gave an excellent overview about 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 made also an introduction into 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) presented new developments in multiplexing and quantitative genotyping of SNPs by minisequencing using microarrays. Prof. Syvänen introduced a four-color tag-microarray system based on cyclic minisequencing reaction in solution using primers carrying 5'tag sequences, followed by capture of the reaction products by hybridization to microarrays carrying complementary tag-oligonucleotides in a "array of arrays" conformation in a pattern compatible with reaction chambers in a 384-microtiter well format.

Markus Beier (Febit AG, Germany) introduced the new geniom® technology developed by Febit AG. The technology represents the first integrated solution of in situ microarray fabrication, hybridization, detection and analysis taking place within one single benchtop instrument. The setup allows for maximum flexibility in design and realization of all kind of genomic assays. He discussed also application of this technology to expression profiling and genotyping experiments.

Anthony J. Brookes (Centre for Genomics and Bioinformatics, Stockholm, Sweden) discussed the advantages of Dynamic Allele-Specific Hybridization (DASH) worked out in his lab, which is based on hybridization of oligonucleotide probe to target DNA, followed by real-time tracking of denaturation events after samples are dynamically heated. They have recently coupled DASH to innovations of centrifugal arrays and iFRET, creating thereby an advanced genotyping platform they termed DASH-2. Prof. Brookes demonstrated also results of population genetic investigations and utility of human haplotype map.

Scott P. White (Center for Human Genome Studies, Bioscience Division, Los Alamos National Lab, Los Alamos, NM, USA) showed results of high-throughput SNP scoring with GAMMArrays and genomic analysis using multiplexed microsphere arrays. The assay uses fluorescently labeled DNA fragments bound to microspheres, which are analyzed using flow cytometry. SNP scoring is performed using minisequencing primers and fluorescently labeled dideoxynucleotides. Multiplexed microspheres allow to score hundreds of SNPs simultaneously.

Niels Storm (Sequenom GmbH, Hamburg, Germany) presented Sequenom's platform for SNP discovery and SNP scoring with MALDI-TOF mass spectrometry. He focused on MassARRAYÔ system combining the advantages of MALDI-TOF with real-time data analysis enabling high-throughput genotyping.

Per Johan Ulfendahl (Pyrosequencing AB, Uppsala, Sweden) gave an overview about PyrosequencingÔ technology and the use of this method for allele frequency quantitation, forensic identification using mitochondrial DNA, detection and typing of bacteria and detection of SNPs in a set of different genes.

Maido Remm (Estonian Biocentre, Tartu, Estonia) introduced software tools for automated selection of SNPs and PCR primer design for large-scale genotyping projects developed at the Estonian Biocentre. Dr. Remm outlined the importance of analyzing all primers against the full genome.

Andres Metspalu (University of Tartu / Estonian Biocentre, Estonia) presented data about large-scale SNP typing using human chromosome 22 as a model. Data from chr 21 and chr 22 LD mapping and modeling have yielded the first generation LD map and allowed to establish the LD block boundaries. The next step will be to type large population samples with minimal number of well-selected SNPs, which are required to reveal the major LD blocks. He also proposed the two-step process for the whole genome LD mapping starting from the exons and expanding to intergenic areas later on.

List of participants ...

A group photo from the meeting ...

Other photos from the meeting are available at the Estonian Biocentre's web site.