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Microarrays: Basic Principle

Microarrays: Basic Principle. Highly parallel molecular search and sort process based on hybridization of nucleic acids. Targets. Probe. A G C C T. A C C G A. T C G G A. C C G G A. G C G G A. Microarray Principles. Probe Array. Targets. Mix Probes and Target:

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Microarrays: Basic Principle

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  1. Microarrays: Basic Principle Highly parallel molecular search and sort process based on hybridization of nucleic acids Targets Probe A G C C T A C C G A T C G G A C C G G A G C G G A

  2. Microarray Principles Probe Array Targets Mix Probes and Target: Selective Hybridization

  3. Microarray Principles Probe Array Targets ~106 probes per spot

  4. Microarray Principles Probe Array GeneExpressionLevel Gene1: 1846 Gene2: 368 …………. …….. …………. …….. Gene10,000: 218

  5. Synthesized High Density Oligonucleotide Chip Design Gene sequence database 5’ 3’ Design a set of short probes for each gene 5’ 3’ Optimize probe choices with BLAST ACGTAC Synthesize probes ACCTAC and controls on the array

  6. Oligonucleotide Array Technology: Chip Design (a) Sequence database Probe for the alpha-myosin gene sequence Synthesize probe on chip GATCCTGACTCTTCTTCAC….. Z20656 GAGAAG probe GAGAAG Synthesize probes Probes for all sequences Optimize Sequence database (b) #!/usr/bin/perl $x=<InputFile1> $x=~s/\/.+?\n/g; Print OutputFile1 “$x”; probe probe probe probe Software to ensure probes are specific to intended target sequences

  7. RNA Extraction, Amplification, Labeling Biotin labeled bases Oligo-dT R-Transcription Amplification Transcription Tissue Total RNA cDNA (from mRNA only) c mRNA (biotin labeled) Fragment, Hybridize, Stain Laser scan

  8. Microarray Processed Output Gene Level Gene1: 1846 Gene2: 368 …………. …….. …………. …….. Gene22,283: 218

  9. Oligonucleotide Array Technology: Experimental Approach cRNA from tissue of interest DNA microarray Image Quantitative gene expression (a) Laser scanning Data processing Hybridization (b) Multiple experiments Pattern discovery Combine, normalize, analyze

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