DNA-MICROARRAY. Katharina Pracht, Mareike Baasch

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1 DN-MIRORRY Katharina Pracht, Mareike Baasch

2 ontent Definition Historical Backround Fundamental concept ypes of DN-Microarrays pplication Experimental procedure (cancer research) Summary Outlook Literature

3 Definition - micro system - analyse thousand of gene in one experiment at the same time - each spot : - contains multiple identical strands of DN - represent one gene - thousand spots are arrayed on solid surface

4 Historical Background 1970 first publication: marked nucleinacids for the analysis of gene expression at solid surface 1993 Bio-hip Project French tomic Energy omission 1995 first DN-Microarrays for the analysis of gene expression (Patrick Braun, Stanford University)

5 Fundamental concept labeled cdn + Hybridisation DN- probe at solid surface

6 ypes of DN-Microarrays glass slides microarrays oligonucleotide chips

7 ypes of Microarrays lass Slides Microarray conditions for glas: inert against solvents mechanical stable small selffluorescense size of 1.3 x 1.3 cm up to clones by slide application: establishment of genexpressionprofils DN probe: off-chip - method

8 ypes of Microarrays off-chip - Method X X X X X X Print cdn hip surface with reactive group X

9 ypes of Microarrays off-chip - Method DN- probes are printed at the solid chip surface by Spotting- needles

10 ypes of Microarrays Oligonucleotid hip enehip (ffymetrix) high density oligonucleotide based DN arrays substrate: quartz wafer oligonucleotides by slide application: - detection / analysis of mutations - gene expression studies DN probe: on-chip - method (in situ)

11 on-chip (in situ) ypes of Microarrays photolithography: O O O O O O mask substrate photolabile protective group covalent linker molecule sequences synthesised directly onto the surface of the substrate at pre-selected positions

12 on-chip (in situ)- Method ypes of Microarrays hν O O O O O O O O O O O H O remove protective group H O O O O O chemical coupling H O O repeat chemical coupling hν remove protective group 15-25mer Oligonucleotide O O O O O O O O

13 pplication fundamental research in molecular biology: gene-expression analysis genotype analysis (mutations) pathogen- and infection-analytics: analysis and identification of microorganism antibiotic resistance human genetic diagnostics: analyse genetic disease (Personal enomics) food and environmental analysis: identification of germ and pathogens identification of genetically modified food (corn, soya) clinical analytics: cancer research effect of drugs (tailormade drugs)

14 pplication What is the difference between a healthy cell and a cancer cell? all cells : copy of every human gene each celltype unique expression profile

15 pplication What is the difference between a healthy cell and a cancer cell? healthy cell on on off gene 1 gene 2 gene 3 mrn mrn cancer cell on off on gene 1 gene 2 gene 3 mrn mrn identify differences in expression profile between healthy cells and cancer cells

16 experimental procedure collect tissue (healthy and cancer cells of the same patient) isolate the mrn make cdn out of mrn, label with different colors (green: healthy cell mrn; red: cancer cell mrn) apply both cdn samples to the same microarray scan microarray, analyse data

17 experimental procedure

18 experimental procedure detection: green scan red scan merged image

19 experimental procedure detection: red spots: turned up in cancer cells green spots: turned down in cancer cells black spots: no cdn hybridized yellow spots: healthy and cancer cell cdn hybridized

20 experimental procedure Problem with yellow spots: healthy cell cancer cell on gene on gene only the mrn is detected mrn mrn protein

21 Summary advantages: time-saving (parallelism) small volume high selectivity re-usable automation disadvantages: technical extensive, expensive development analyse only known genes depending on chipproducers to expensive for every day use in laboratory minimisation

22 Outlook efficient application in molecular diagnostics: infection disease genetic disease drug development simple and cost- effectiv production clinical microbiologie

23 Literature root%2ftutech.tutech.net%2fpdfs&field=rw&filename=perspektiven_microarrays_kroll.pdf&c ontenttype=application/pdf

24 hanks for your ttention

25 Zusammenfassung 1. Was ist ein DN- hip? Mikrosystem, das es ermöglicht tausende ene gleichzeitig zu untersuchen. DN- Sonden, die jeweils ein en repräsentieren, sind dabei an eine feste Oberfläche gebunden. 2. Welche rten werden heute kommerziell genutzt? las DN- hips Oligonukleotid hips 3. Synthese der DN- Sonden: off hip : bereits synthetisierte cdn oder PR Fragmente werden an die Oberfläche gebunden on hip : Oligonukleotide werden Stück für Stück auf den hip gebracht

26 Zusammenfassung 4. Detektion rote Punkte: ene die nur in der Krebszelle exprimiert werden. grüne Punkte: ene die nur in der gesunden Zelle exprimiert werden. schwarze Punkte: ene die weder in der gesunden, noch in der Krebszelle exprimiert werden und damit uninteressant sind. gelbe Punkte: ene die sowohl in der gesunden, als auch in der Krebszelle exprimiert werden. Da nur die mrn detektiert wird, können diese unter Umständen (kleinere Mutationen die zu keiner Proteinsynthese führen) trotzdem interessant sein.