Le dogme central de la biologie moléculaire

Présentation au sujet: "Le dogme central de la biologie moléculaire"— Transcription de la présentation:

1 Le dogme central de la biologie moléculaire
ADN ARNm Protéine

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5 Les puces à ADN : principe

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

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9 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

10 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

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

15 Colorimétrie …

16 Colorimétrie

17 Radioactivité … Clontech, Nylon

18 Cellules Leucemiques Cellules Normales

19 fluorescence … Puce Affymetrix

20 fluorescence … Puce Affymetrix

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22 Exemple d’application

23 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

24 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%)

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

26 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

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

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

29 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

30 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

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

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

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

34 Les scanners

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