Microarrays

Curso Dr. Alvarez

1. DNA MICROARRAYS

2. Microarrays: Universal Biochemistry Platforms 2 Peptides Proteins Carbohydrates Lipids Small molecules DNA

3. Some questions for the golden age of genomics How gene expression differs in different cell types? How gene expression differs in a normal and diseased (e.g., cancerous) cell? How gene expression changes when a cell is treated by a drug? How gene expression changes when the organism develops and cells are differentiating? How gene expression is regulated – which genes regulate which and how? 3

4. What is a DNA Microarray? Biological Samples in 2D Arrays on Membranes or Glass Slides 4 Cheung et al. 1999

5. What is a DNA Microarray? Also known as DNA Chip Allows simultaneous measurement of the level of transcription for every gene in a genome (gene expression) Microarray detects mRNA, or rather the more stable cDNA 5

6. MICROARRAY TYPES 6

7. The Colours of a Microarray GREEN represents Control DNA , where either DNA or cDNA derived from normal tissue is hybridized to the target DNA. RED represents Sample DNA , where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA. YELLOW represents a combination of Control and Sample DNA , where both hybridized equally to the target DNA. BLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA. 7

8. Microarray Steps Experiment and Data Acquisition Sample preparation and labeling Hybridization Washing Image acquisition Data normalization Data analysis Biological interpretation 8

9. I. Target and probe preparation 9

10. There are many ways to obtain a labeled target sample. 10 ...GGCUUAAUGAGCCUUAAAAAA...A mRNA TTTTTT...T viral enzyme reverse transcriptase recognizes poly-T bound to poly-A and begins to add complementary DNA nucleotides. The C nucleotides are dyed. A A A G G C T C T T A A G C C ... poly-A tail poly-T primer cDNA target

11. Hybridization and Data Analysis 11

12. Spotted Array Synthesis. 12

13. Microarray Experiment 13

14. How do we manufacture a microarray? Start with individual genes, e.g. the ~6,200 genes of the yeast genome Amplify all of them using polymerase chain reaction (PCR) “Spot” them on a medium, e.g. an ordinary glass microscope slide Each spot is about 100 µm in diameter Spotting is done by a robot Complex and potentially expensive task 14

15. Robotic spotting 15

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17. DNA Samples on 96 well plates 17

18. 18 18 The PixSys 5500 Arraying Robot (Cartesian Technologies) Vacuum wash station The print head holds up to 32 pins in a 8x4 format Vacuum hold-down platform (50 slide capacity) Robotic arm

19. Contact Printing 19

20. Non Contact Printing 20 InkJet (HP/Canon) technology • 1 drop = 100 picolitres

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22. 22 Spotting the Probes on the Microarray 8 X 4 Print Head microarray slide plate with wells holding probes in solution All spots of the same color are made at the same time. All spots in the same sector are made by the same pin.

23. Using cDNA Microarrays to Measure mRNA Levels 23 ACCTG...G ACCTG...G ACCTG...G TTCTG...A TTCTG...A TTCTG...A GGCTT...C GGCTT...C GGCTT...C ATCTA...A ATCTA...A ATCTA...A ACGGG...T ACGGG...T ACGGG...T CGATA...G CGATA...G CGATA...G ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? Sample 1 Sample 2 Microarray Slide Spots (Probes) Unknown mRNA Sequences (Target)

24. Extract mRNA 24 ACCTG...G ACCTG...G ACCTG...G TTCTG...A TTCTG...A TTCTG...A GGCTT...C GGCTT...C GGCTT...C ATCTA...A ATCTA...A ATCTA...A ACGGG...T ACGGG...T ACGGG...T CGATA...G CGATA...G CGATA...G ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? Sample 1 Sample 2

25. Convert to cDNA and Label with Fluorescent Dyes 25 ACCTG...G ACCTG...G ACCTG...G TTCTG...A TTCTG...A TTCTG...A GGCTT...C GGCTT...C GGCTT...C ATCTA...A ATCTA...A ATCTA...A ACGGG...T ACGGG...T ACGGG...T CGATA...G CGATA...G CGATA...G Sample 1 Sample 2 ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? Sample 1 Sample 2

26. Mix Labeled cDNA 26 ACCTG...G ACCTG...G ACCTG...G TTCTG...A TTCTG...A TTCTG...A GGCTT...C GGCTT...C GGCTT...C ATCTA...A ATCTA...A ATCTA...A ACGGG...T ACGGG...T ACGGG...T CGATA...G CGATA...G CGATA...G Sample 1 Sample 2 ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ??????????

27. Hybridize cDNA to the Slide 27 ACCTG...G ACCTG...G ACCTG...G TTCTG...A TTCTG...A TTCTG...A GGCTT...C GGCTT...C GGCTT...C ATCTA...A ATCTA...A ATCTA...A ACGGG...T ACGGG...T ACGGG...T CGATA...G CGATA...G CGATA...G Sample 1 Sample 2 ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ??????????

28. Excite Dyes with Laser 28 ACCTG...G ACCTG...G ACCTG...G TTCTG...A TTCTG...A TTCTG...A GGCTT...C GGCTT...C GGCTT...C ATCTA...A ATCTA...A ATCTA...A ACGGG...T ACGGG...T ACGGG...T CGATA...G CGATA...G CGATA...G Sample 1 Sample 2 ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ??????????

29. Scan 29 ACCTG...G ACCTG...G ACCTG...G TTCTG...A TTCTG...A TTCTG...A GGCTT...C GGCTT...C GGCTT...C ATCTA...A ATCTA...A ATCTA...A ACGGG...T ACGGG...T ACGGG...T CGATA...G CGATA...G CGATA...G Sample 1 Sample 2 ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ?????????? ??????????

30. Quantify Signals 30 ACCTG...G 7652 138 TTCTG...A 5708 4388 GGCTT...C 8566 765 ATCTA...A 1208 13442 ACGGG...T 6784 9762 CGATA...G 67 239 Sample 1 Sample 2

31. Patrick Schmid 31

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34. Oligonucleotide Microarray Gene chip (DNA chip, Affymetrix chip): Oligonucleotide (20~80-mer oligos) is synthesized either in situ (on-chip) Developed at Affymetrix, Inc. , under the GeneChip® trademark 34

35. Affymetrix Chip Each gene has 16 – 20 pairs of probes synthesized on the chip Each pairs of probes have two oligonucleotide – Perfect match (PM, reference seq) ATG…C…TGC (20-25 bases) – Mismatch (MM, one base change) ATG… T …TGC A MM oligo is identical to a PM oligo except that the middle nucleotide (13 th of 25) is intentionally replaced by its complementary nucleotide . The scanned result for a given gene is the average differences between PM and MM signals, over probes 35

36. Different Probe Pairs Represent Different Parts of the Same Gene 36 gene sequence Probes are selected to be specific to the target gene and have good hybridization characteristics.

37. A Probe Set for Measuring Expression Level of a Particular Gene 37 probe pair probe cell gene sequence ...TGCAATGGGTCAGAA G GACTCCTATGTGCCT... AATGGGTCAGAA G GACTCCTATGTG AATGGGTCAGAA C GACTCCTATGTG perfect match sequence mismatch sequence probe set

38. Affymetrix Chip 38

39. The photolithographic method Treat substrate with chemically protected “linker” molecules, creating rectangular array Selectively expose array sites to light deprotects exposed molecules, activating further synthesis Flush chip surface with solution of protected A,C,G,T Binding occurs at previously deprotected sites Repeat steps 2&3 until desired probes are synthesized 39

40. Photolithography 40 The mask only allows light to pass to specific features on the chip

41. Photolithography 41

42. Affymetrix chip Photolithographic Approach In-situ synthesis of oligonucleotide 42

43. Photolithographic Approach 43

44. Patrick Schmid 44 Affymetrix Arrays

45. Affymetrix GeneChips 45 The black features represent no intensity (no RNA hybridized to the respective probe in the feature). The intensity level from lowest to highest by color is: Dark blue -> Blue -> Light Blue -> Green -> Yellow -> Orange -> Red - > White . More intensity means more RNA bound to a specific feature, which basically means the gene was expressed at a higher level.

46. Affymetrix GeneChip experiment 46

47. Affymetrix GeneChip experiment labeled cRNA randomly fragmented in to pieces anywhere from 30 to 400 base pairs in length The fragmented, Biotin-labeled cRNA is added to the array Anywhere on the array where a RNA fragment and a probe are complimentary, the RNA hybridizes to the probes in the feature. The array is then washed to remove any RNA that is not stuck to an array then stained with the fluorescent molecule that sticks to Biotin (Cy5 conjugated to streptavidin) Lastly, the entire array is scanned with a laser and the information is kept in a computer for quantitative analysis of what genes were expressed and at what approximate level 47

48. in-situ synthesized arrays The different methods for deprotection lead to the three main technologies for making in-situ synthesized arrays: Photodeprotection using masks: this is the basis of the Affymetrix ® technology. Photodeprotection without masks : this is the method used by Nimblegen and Febit . Chemical deprotection with synthesis via inkjet technology: this is the method used by Rosetta, Agilent and Oxford Gene Technology. 48

49. Photodeprotection without masks 49

50. Maskless Array Synthesis 50

51. 51 NimbleGen Arrays

52. Microarray Experiment 52

53. Cancer and Microarrays 53

54. * Measuring levels of gene expression * Creating diagnostic tests to predict whether a patient has a genetic predisposition to obesity * Designing Drugs 54 Gene expression and obesity

55. Reading an array (cont.) Block Column Row Gene Name Red Green Red:Green Ratio 1 1 1 tub1 2,345 2,467 0.95 1 1 2 tub2 3,589 2,158 1.66 1 1 3 sec1 4,109 1,469 2.80 1 1 4 sec2 1,500 3,589 0.42 1 1 5 sec3 1,246 1,258 0.99 1 1 6 act1 1,937 2,104 0.92 1 1 7 act2 2,561 1,562 1.64 1 1 8 fus1 2,962 3,012 0.98 1 1 9 idp2 3,585 1,209 2.97 1 1 10 idp1 2,796 1,005 2.78 1 1 11 idh1 2,170 4,245 0.51 1 1 12 idh2 1,896 2,996 0.63 1 1 13 erd1 1,023 3,354 0.31 1 1 14 erd2 1,698 2,896 0.59 55 Campbell & Heyer, 2003

56. Color Coding Tables are difficult to read Data is presented with a color scale Coding scheme: Green = repressed (less mRNA) gene in experiment Red = induced (more mRNA) gene in experiment Black = no change (1:1 ratio) Or Green = control condition (e.g. aerobic) Red = experimental condition (e.g. anaerobic) We only use ratio 56 Campbell & Heyer, 2003

57. Clustering of example 57 Campbell & Heyer, 2003

58. Clustering of entire yeast genome 58 Campbell & Heyer, 2003

59. SMD Database 59

60. Microarray software 60

61. Microarray databases and tools 61

62. Microarray tools NetAffix Analysis center from affymetrix Array content information Probe sequences Gene annotations Xcluster- tool for cluster analysis GENECLUSTER TIGR Microarray MADAM- Microarray data manager SPOTFINDER -image processing tool MIDAS -Microarray data analysis system MEV -MultiExperiment Viewer 62

63. ARRAY EXPRESS (EBI) 63

64. GEO(NCBI) 64