TRANSCRIPTOME 2002: From Functional Genomics to Systems
Microarray Technology for Transcript Profiling and Analysis of Genetic Variability
Joakim Lundeberg, Department of Biotechnology, Royal Institute of Technology, Stockholm, SWEDEN
Microarray systems for analysis of transcript profiles and genetic variability have been established to elucidate and map gene function in different biological systems. Transcript profiles can be achieved by subtractive or by global methods such as SAGE, EST sequencing or array based technologies. Here we will give an example of the combined use of subtractive methods and global cDNA arrays to identify low abundant transcripts. Furthermore systems for achieving high quality cDNA arrays and procedures to facilitate analysis of minute amounts of samples will be described. The former relates to that it has been shown that relatively high concentrations of probe nucleic acids are required to yield a high dynamic range for the subsequent hybridisation assays. Here we present a novel approach to capture and isolate of PCR products in conditions suitable for printing cDNA slides. We have also developed a microarray based allele specific extension method for the accurate typing of SNPs involving fluorescent nucleotides. The use of allele-specific primers has previously been employed to identify single base variations but it is acknowledged that certain mismatches are not refractory to extension thereby reducing the discriminatory power of these extension assays. Here we have overcome this limitation by introducing apyrase, a nucleotide degrading enzyme, to the extension reaction. Examples will given including analysis of cardiovascular SNP markers as well scanning analysis of whole gene sequences.
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