Systems_Biology_2009_Lesson_4

Systems_Biology_2009_Lesson_4 - Systems Biology Prof....

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Unformatted text preview: Systems Biology Prof. Hilson Academic year 2009 - 2010 1 Systems Biology Systems Biology 4. Array . Array-based based nucleid nucleid acid analysis acid analysis Prof. Prof. Pierre Hilson Pierre Hilson VIB Department of Plant Systems Biology VIB Department of Plant Systems Biology University of Ghent University of Ghent Academic year Academic year 2009 2009-2010 2010- Array types Outline- Spotted arrays- Oligo arrays- Tiling arrays- Array hybridization- Genome annotation with microarrays- Copy number variant analysis Systems Biology Prof. Hilson Academic year 2009 - 2010 2 Array Array-based DNA Analysis based DNA Analysis ¤ Based on the principle that – every nucleic acid strand carries the capacity to recognize complementary sequences through base pairing – Widely used to monitor individual sequences in complex mixtures: Southern (DNA) and Northern (RNA) hybridization ¤ Approach for highly parallel analysis – Many sequences in complex mixtures can be interrogated simultaneously • Low density - Macro arrays – DNA spotted on membrane filters • High density - Microarrays – using glass surfaces ¤ Two types of arrays S th ti ( li l tid ) s – Synthetic (oligonucleotide) arrays – Spotted (DNA) arrays Principal Types of Arrays Principal Types of Arrays ¤ Spotted arrays – Produced by robotic deposition of nucleic acids (PCR products, plasmids or oligonucleotides) onto a glass slide ¤ Oligonucleotide arrays – Produced by in situ synthesis, of short 25-70 mer oligonucleotides onto glass slides Reprinted from: Lipshutz et. al., Nature Genet. 21, 20 (1999) Systems Biology Prof. Hilson Academic year 2009 - 2010 3 Spotted Microarrays Spotted Microarrays ¤ Deposition of nanoliter micro droplets on glass surfaces – Multi-pin tools transfer liquid from micro titer plates on – Chemically treated glass surface • Chemical coating for covalent binding nucleic acid Chemical coating for covalent binding nucleic acids – Moderate densities: ~ 25.000 to 50.000 probes per slide DNA spotting DNA spotting Prehybridization Blocking Prehybridization Blocking Hybridization Hybridization Silanized Slides Silanized Slides Washing Washing Transcribe RNA to Transcribe RNA to labeled cDNA labeled cDNA Complex DNA Production Schemes Complex DNA Production Schemes Master stock Master stock Glycerol Stocks of UniGem Glycerol Stocks of UniGem clones clones Plasmid prep, Millipore Plasmid prep, Millipore 1ml Culture 1ml Culture 2x 100μl PCR 2x 100μl PCR PCR purified Millipore PCR purified Millipore Work stock Work stock PCR purified, Millipore PCR purified, Millipore 384 well spot plate 384 well spot plate Short term stock Short term stock Analysis of quality and yield Analysis of quality and yield using Gel Electrophoresis using Gel Electrophoresis Systems Biology Prof. Hilson Academic year 2009 - 2010 4 Spotted Spotted Microarray applications Microarray applications ¤ Expression microarrays – Used to monitor gene expression • one DNA (PCR) probe is used for each gene...
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Systems_Biology_2009_Lesson_4 - Systems Biology Prof....

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