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Selection of optimal oligonucleotide probes for microarrays using 利用寡核苷酸探针的微阵列的最佳选择
Selection of optimal oligonucleotide probes for microarrays using multiple criteria, global alignment and parameter estimation Xingyuan Li, Zhili He1 and Jizhong Zhou1. 6114–6123 Nucleic Acids Research, 2005, Vol. 33, No. 19. cDNA Microarray: NIEHS Tox Chip GeneChip? Probe Arrays GeneChip? Probe Arrays Multiple Specific Probe Pairs per Gene Detection of genes using Oligos Synthesis of Ordered Oligonucleotide Arrays What’s the complexity? More genes More information per experiment Qualitative and Quantitative Scoring – importance of probe design Procedures for Target Preparation Expression Analysis Hybridization and Staining Creating cRNA from Original RNA Sample Isolated mRNA is reverse transcribed into cDNA using a T7-primer, which contains a poly-T site to bind and select mRNA for amplification. E. coli RNase H digests the original RNA, leaving the cDNA behind. E. coli Polymerase I is added to synthesize a complimentary strand of cDNA. The two strands of cDNA are denatured and T7-RNA polymerase transcribes cRNA while incorporating biotinylated nucleotide bases. Why So Many Probe Pairs? Point Mutations, Deletions, or Insertions will not effect the detection of the gene of interest. Bioinformatics algorithm will account for expression across 11 different probe pairs to calculate expression of gene. Redundancy of probe synthesis Multiple Indicators for the Same Gene Ensures: Quantitative accuracy High sensitivity Indicators of oligonucleotide Specificity: Sequence identity to non-targets Continuous stretch to non-targets Free energy of Binding to the non-targets All these 3 criteria important for the selection of optimal probes Problems with probe synthesis – addressed by CommOligo Representation of each sequence in a genome wide search Liberal cut-offs and fewer non specifics Generally use BLAST for local alignment or Suffix arrays for exact string search Homologous sequence studies versus whole genome arrays ? Applicability to experiments Experiment
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