Le dogme central de la biologie moléculaire ADN ARNm Protéine

Les puces à ADN : principe

Puce à ADN Macroarrays : 2 à 500 sondes Microarrays : 500 à 10.000 sondes Oligo chips : jusqu’a 100.000 sondes Les Supports Nylon Verre/Silice Les sondes Plasmides Produits PCR Oligonucléotides (entre 18 et 70 pb)

Synthesis of Ordered Oligonucleotide Arrays ADRESSAGE PHOTOCHIMIQUE Synthesis of Ordered Oligonucleotide Arrays HO HO O O O Mask Light (deprotection) T T O O O T – HO HO O O O O O O O O Light removes protecting groups at defined positions. Single nucleotide washed over the chip, binds where the protecting group removed. Through successive steps, any sequence can be built up in any position on the chip. The number of steps corresponds with length of oligo, so can increase # of genes without # of steps Substrate

Synthesis of Ordered Oligonucleotide Arrays ADRESSAGE PHOTOCHIMIQUE Synthesis of Ordered Oligonucleotide Arrays Light (deprotection) Mask O O O O O HO HO O O O T T O O O T – Substrate Light removes protecting groups at defined positions. Single nucleotide washed over the chip, binds where the protecting group removed. Through successive steps, any sequence can be built up in any position on the chip. The number of steps corresponds with length of oligo, so can increase # of genes without # of steps Light (deprotection) Mask C A T A T A G C T G T T O O O T T C C O T T C C G C – Substrate REPEAT

Marquage de la Cible Colorimétrie cinétique enzymatique mal connue Fluorescence autofluorescence du Nylon interférence avec le Nylon Radioactivité 32P ou 33P radioactif !!

Colorimétrie …

Colorimétrie

Radioactivité … Clontech, Nylon

Cellules Leucemiques Cellules Normales

fluorescence … Puce Affymetrix

fluorescence … Puce Affymetrix

Exemple d’application

Principle of a DNA array production SAGE data Primers design and synthesis PCR products Purification Robotic deposition on glass slides Scan Analysis Normal cells cDNA labeled with Cy3 or Cy5 Tumoral cells

Thematic DNA array : monocytic differentiation SAGE data Primers design and synthesis PCR products Purification Robotic deposition on glass slides Control of PCR products on agarose gel (1%)

Thematic DNA array : monocytic differentiation SAGE data Millipore purification system Primers design and synthesis PCR products Purification Robotic deposition on glass slides

Thematic DNA array : monocytic differentiation SAGE data Primers design and synthesis All genes spotted in duplicates Corning glass slides (amine chemistry) PCR products Purification Robotic deposition on glass slides

Thematic DNA array : monocytic differentiation Eurogridder ChipWriter Pro (Virtek, ESI) SAGE data Primers design and synthesis PCR products Purification Robotic deposition on glass slides

DNA spotting technology (Pat Brown mean) steel spotting pin chemically modified slides 1 nanoliter spots 120-150 µm diameter 384 well source plate

Thematic DNA array : monocytic differentiation + + + + + - Amine slide Slide processing Slide quality control UV crosslink and oven baking SDS washing succinic anhydride/ N-methyl pyrrolidone / Na borate treatment denaturation RNA labeling Hybridization

Quality control of array production Slide processing Principle: fluorescent nucleotides are added to the end of the spotted fragments by the enzyme terminal transferase (Roche kit) Slide quality control RNA labeling Hybridization

Thematic DNA array : monocytic differentiation Slide processing blablablabla Slide quality control RNA labeling blablablabla Hybridization Hybridisation ON, 42°C, volume : 15µl

Thematic DNA array : monocytic differentiation Slide processing cDNA Cy3/5 Slide Slide quality control RNA labeling Hybridization Corning hybridization chamber

Thematic DNA array : monocytic differentiation Slide processing Slide quality control RNA labeling Hybridization

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