What is a microarray

Transcription

1 DNA Microarrays

2 What is a microarray A surface on which sequences from thousands of different genes are covalently attached to fixed locations (probes). Glass slides Silicon chips Utilize the selective nature of DNA-DNA or DNA-RNA hybridization

3 Types of microarrays Probe Manufacture Spotted Arrays DNA probes are synthesized and then spotted onto the microarray Customized for each experiment in-lab Low cost Oligonucleotide microarrays DNA oligonucleotides are synthezised directly on the microarray Commercial arrays (Affymetrix, Nimblegen and Combimatrix) expensive Detection Two-color detection Spotted Array: Compare two samples by labeling with two different flurophores and analyzing on the same array Cy5 (red) and Cy3 (green) One-color detection Determine gene expression level One array per sample

4 Phosphoramidite chemistry Stable = can actually be shipped High coupling yield Synthesis on a solid support Monomer addition Still a manual method, not consistent Faster cycle time of 20 minutes Using some nasty reagents (e.g. DMAP, DMF, Thiophenol) Very cyclical and repetitive = not fun

5 Coupling efficiency 100 % 50 % Phosphoramidite chemistry at 98% coupling efficiency 0 % 10 bases 20 bases

6 Solid Phase Synthesis Controlled Pore Glass (CPG) support covalently linked with one of the four nucleosides Reactive groups of nucleosides are blocked or protected to prevent unwanted side reactions DNA synthesis occurs by connecting nucleoside monomers one at a time to the 5' end of growing chain (3' to 5' end) DNA chain is cleaved from the support by ammonia treatment

7 Solid Phase Synthesis CPG support covalently linked with one of the four nucleosides

8 DMT - protected nucleoside CH 3 O DMT group C O BASE CPG O Linker O O CH 3 C O CH 2 CH 2 C O NH CH CH CH S i O O

9 Solid Phase Synthesis CPG support covalently linked with one of the four nucleosides Reactive groups of nucleosides are blocked or protected to prevent unwanted side reactions

10 Protection of Base Amino Groups O N O NH C N NH HN N C O N N N N CH CH 3 CH 3 Adenosine - benzoyl protected Guanosine - isobutyryl protected O O NH C HN CH 3 N O N O N Thymidine Cytidine - benzoyl protected

11 Solid Phase Synthesis CPG support covalently linked with one of the four nucleosides Reactive groups of nucleosides are blocked or protected to prevent unwanted side reactions DNA synthesis occurs by connecting nucleoside monomers one at a time to the 5' end of growing chain (3' to 5' end)

12 Direction of synthesis Oligonucleotide 5 Natural DNA CPG

13 Solid Phase Synthesis CPG support covalently linked with one of the four nucleosides Reactive groups of nucleosides are blocked or protected to prevent unwanted side reactions DNA synthesis occurs by connecting nucleoside monomers one at a time to the 5' end of growing chain (3' to 5' end) DNA chain is cleaved from the support by ammonia treatment

14 Typical quantities of oligonucleotide obtained from different scales Synthesis scale Crude Yield (O.D.) (20 mers) Primer (40 nmole) 0.2 umole 1 umole 10 umole

15 Analysis & Purification PAGE HPLC CE (capillary electrophoresis) OPC (oligonucleotide Purification Cartridge)

16 Quantitation & Storage UV spectroscopy: Measuring OD at 260 nm 1 OD 260 for ssdna = 33 ug/ml Storage: stable with little or no degradation for long periods of time (over a year)

17 Alternative chemistries Antisense (phosphorothioate) RNA oligoribonucleotides (using 2' silyl 5' DMT-CE phosphoramidite RNA monomers) 5' modified oligonucleotides

18 5' modified oligonucleotides Aminolink Enzyme - labeled Biotin - labeled Fluorescein-lebeled

19 Applications of Oligonucleotides Probes Primers Synthetic gene Mutagenesis

20 Microarray Platforms! " Spotted" DNA fragments (usually created by PCR)or oligos are stuck to glass slides" The size of the fragment can be any length (usually 500 bp-1 kb)" The size of the oligos range from nts" These arrays can be created in individual labs using affordable equipment" " " Affymetrix" Affymetrix arrays are typically limited to oligos of nts" The probes on these arrays are synthesized using a light mask technology " Photo-sensitive reactions are used to remove a blocking group and then extend " It is very costly to fabricate masks for a new array design " Not commonly used for custom arrays" " "

21 Types of Microarray Platforms! NimbleGen Maskless Array Synthesizer technology uses PowerPoint projector parts; 786,000 tiny aluminum mirrors are used to shine light in specific patterns, Photo deposition chemistry allows single nt extensions. 380,000 or 2.1 million oligos/array (50mers). Agilent Uses ink-jet printer technology. Grows another base at the end of a molecule by deprotecting the end and attaching a new base with a protected end to avoid duplication. 244,000 oligos/array (60mers). Combimatrix Semiconductor technology directs the assembly of a specific sequence of DNA bases in response to a digital command. Each feature on the array (a microelectrode) is digitally addressed to controls the addition of a new base. 12,000 oligos (50mers)/array; 40,000 feature arrays in development.

22 Making a Spotted Arrays Probes cdna microarrays (up to 3000 bp) Long-oligonucleotide spotted arrays: uniform length ( bp) Customized spotted array Spotting pins draw fluid (containing DNA probes) by capillary action and form spots on the side through surface tension interaction between the surface and spotting buffer. spotted onto glass slide using robotic arms Commercial spotted arrays (Agilent) SurePrint Technology Iike an inkjet printer, prints sets of cdna clones or oligonucleotides one-by-one

23 Spotted Arrays: Sample Preparation and Two-color Detection Allows a direct comparison between two different samples

24 DNA microarrays can be manufactured by: Photolitography (Affymetrix, Nimblegen) Inkjet (Agilent, Canon) Robot spotting (many providers)

25 Affymetrix photolitography Each probe 25 bp long probes per gene Perfect Match (PM) as well as MisMatch (MM) probes

26 Masked Array Synthesis (Affymetrix)

27 Maskless Arrays (Nimblegen)

28 NimbleGen photolitography

29 Robot Spotting

30 InkJet (HP/Canon) technology

31 Making a Oligonucleotide array Probes bp oligonucleotide probes Electrochemistry - CombiMatrix Array Each microelectrode selectively generates chemical reagents by electrochemical reaction. Controls the building of DNA on a semiconductor chip by the software-controled turning on and off of electrodes. Photolithography - Affymetrix Array manipulates light to direct the chemical synthesis of the probes Mask directs the flow of U.V. light Uses light sensitive protecting groups.

32 Applications Expression profiling Comparative genomic hybridization Single nucleotide polymorphism Sequencing

33 Summary

34

35 References GeneChip Expression Analysis Technical Manual. support/downloads/manuals/expression_analysis_technical_manual.pdf. McInnerney Kate. One-cycle eukaryotic target prep protocol. Agilent SurePrint Technology (2003) Retrieved March 23, 2008 from Spotted Array Printing Techniques NCBI A Science Primer: Microarrays: Chipping away at the myteries of science and medicine. Affymetrix GeneChip Workflow tools Affymetrix Educator Resources

36 Image Analysis 1. Gridding: identify spots (automatic, semiautomatic, manual) 2. Segmentation: separate spots from background. Fixed circle (B), Adaptive circle C, Adaptive shape (D), Histogram 3. Intensity extraction: mean or median of pixels in spot 4. Background correction: local or global

37 Distribution of mrna levels in different cells

38

39 Photolithography

40 Removal of protecting groups and caps

41 DNA Synthesis Chemistry Cycle Detritylation Coupling Capping Oxidation

42 Oligonucleotide Array: Eukaryotic Target Preparation and Onecolor detection

43 Detection of mrna