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DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or...

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DNA Microarrays
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Page 1: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

DNA Microarrays

Page 2: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 3: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 4: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 5: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Coupling efficiency

100 %

50 %

0 %

10 bases 20 bases

Phosphoramidite chemistry at 98% coupling efficiency

Page 6: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 7: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Solid Phase Synthesis

CPG support covalently linked with one of the four nucleosides

Page 8: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

DMT - protected nucleoside

CH 2 O

CH 2 C O

NH CH 2 CH

2 CH 2 Si

O O C

O

O

BASE O C

O

CH 3

O

CH 3

Page 9: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 10: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Protection of Base Amino Groups

NH

N N

N O

HN C O CH

CH 3 CH 3

HN N

O

O

CH 3

N N

NH

O

C O

N N N

N NH C

O

Page 11: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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)

Page 12: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Direction of synthesis

CPG 3’5’

Oligonucleotide

Natural DNA 3’5’

Page 13: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 14: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

20 - 25

5 - 10

100 - 120

800 - 1000

Page 15: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Analysis & Purification

PAGE

HPLC

CE (capillary electrophoresis)

OPC (oligonucleotide Purification Cartridge)

Page 16: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Quantitation & Storage

Storage: stable with little or no degradation for

long periods of time (over a year)

UV spectroscopy: Measuring OD at 260 nm

1 OD 260 for ssDNA = 33 ug/ml

Page 17: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Alternative chemistries

5' modified oligonucleotides

RNA oligoribonucleotides (using 2' silyl 5' DMT-CE phosphoramidite RNA monomers)

Antisense (phosphorothioate)

Page 18: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

5' modified oligonucleotides

Aminolink

Enzyme - labeled

Biotin - labeled

Fluorescein-lebeled

Page 19: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two
Page 20: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Microarray Platforms

Page 21: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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.

Types of Microarray Platforms

Page 22: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 23: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Allows a direct comparison between two different samples

Spotted Arrays: Sample Preparation and Two-color Detection

Page 24: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 25: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

well as MisMatch (MM) probes

Page 26: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Masked Array Synthesis (Affymetrix)

Page 27: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Maskless Arrays (Nimblegen)

Page 28: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

NimbleGen photolitography

Page 29: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Robot Spotting

Page 30: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

InkJet (HP/Canon) technology

Page 31: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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.

Page 32: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Applications   Expression profiling   Comparative genomic hybridization   Single nucleotide polymorphism   Sequencing

Page 33: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Summary

Page 34: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two
Page 35: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 36: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

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

Page 37: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Distribution of mRNA levels in different cells

Page 38: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two
Page 39: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Photolithography

Page 40: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Removal of protecting groups and caps

Page 41: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

DNA Synthesis Chemistry Cycle

Detritylation

Coupling

Capping Oxidation

Page 42: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Oligonucleotide Array: Eukaryotic Target Preparation and One-color detection

Page 43: DNA Microarrays - Courses · PDF fileDNA Microarrays . What is a microarray ... of DNA-DNA or DNA-RNA hybridization . ... Spotted Array: Compare two

Detection of mRNA


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