What is a microarray

DNA Microarrays What is a microarray A surface on which sequences from thousands of different genes are covalently attached to fixed locations (prob...
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DNA Microarrays

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

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

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

Coupling efficiency 100 %

50 %

Phosphoramidite chemistry at 98% coupling efficiency

0% 10 bases

20 bases

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

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

DMT - protected nucleoside CH 3 O

C

O

O O CH3

BASE

O

C CH CH2 C NH CH CH CH S O 2 2 2 2 i O O O

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

Protection of Base Amino Groups O O NH C

HN

N C O

N

N

CH CH3 CH3

N

N

N

NH

O O CH 3

HN O

NH C

N

N O

N

N

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)

Direction of synthesis Oligonucleotide 5’

3’

Natural DNA 5’

3’

CPG

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

Typical quantities of oligonucleotide obtained from different scales Synthesis scale

Crude Yield (O.D.) (20 mers)

Primer (40 nmole)

5 - 10

0.2 umole

20 - 25

1 umole

100 - 120

10 umole

800 - 1000

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

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)

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

5' modified oligonucleotides

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

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 20-100 nts These arrays can be created in individual labs using “affordable” equipment

Affymetrix Affymetrix arrays are typically limited to oligos of 20-25 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

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.

Making a Spotted Arrays Probes   cDNA microarrays (up to 3000 bp)   Long-oligonucleotide spotted arrays: uniform length (20 -100 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

Spotted Arrays: Sample Preparation and Two-color Detection

Allows a direct comparison between two different samples

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

Affymetrix photolitography •  Each probe 25 bp long •  22-40 probes per gene •  Perfect Match (PM) as well as MisMatch (MM) probes

Masked Array Synthesis (Affymetrix)

Maskless Arrays (Nimblegen)

NimbleGen photolitography

Robot Spotting

InkJet (HP/Canon) technology

Making a Oligonucleotide array Probes   20-100 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.

Applications   Expression profiling   Comparative genomic hybridization   Single nucleotide polymorphism

  Sequencing

Summary

References  

GeneChip Expression Analysis Technical Manual. http://www.affymetrix.com/ support/downloads/manuals/expression_analysis_technical_manual.pdf.

 

McInnerney Kate. One-cycle eukaryotic target prep protocol. http://www.homepage.montana.edu/~kmcinner/Affyinfo.html

 

Agilent SurePrint Technology (2003) Retrieved March 23, 2008 from http://www.chem.agilent.com/temp/radD1958/00000176.PDF

 

Spotted Array Printing Techniques http://www.flychip.org.uk/printingintroduction.php

 

NCBI A Science Primer: Microarrays: Chipping away at the myteries of science and medicine. www.ncbi.nlm.nih.gov/About/primer/microarrays.html

 

Affymetrix GeneChip Workflow tools http://www.invitrogen.com/content.cfm?pageid=11455

 

Affymetrix Educator Resources http://affymetrix.com/corporate/outreach/educator.affx

Image Analysis 1.  2. 

3.  4. 

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

Distribution of mRNA levels in different cells

Photolithography

Removal of protecting groups and caps

DNA Synthesis Chemistry Cycle Detritylation Coupling Capping Oxidation

Oligonucleotide Array: Eukaryotic Target Preparation and Onecolor detection

Detection of mRNA