DNA chips

Technologies and utility

Historical perspective
 DNA hybridization (1960s)
 Detection of hybrids
 hydroxyapatite
 radioactive labelling
 enzyme-linked detection
 fluorescent labelling
 Fixing sample on solid support
 Southern blots (1970s)
 Northern blots
 Dot blots
Basic principles
 Main novelty is one of scale
 hundreds or thousands of probes rather than
tens
 Probes are attached to solid supports
 Robotics are used extensively
 Informatics is a central component at all
stages
Major technologies
 cDNA probes (> 200 nt), usually produced
by PCR, attached to either nylon or glass
supports
 Oligonucleotides (25-80 nt) attached to
glass support
 Oligonucleotides (25-30 nt) synthesized in
situ on silica wafers (Affymetrix)
 Probes attached to tagged beads
Principal uses of chips
 Genome-scale gene expression analysis
 Differentiation
 Responses to environmental factors
 Disease processes
 Effects of drugs
 Detection of sequence variation
 Genetic typing
 Detection of somatic mutations (e.g. in oncogenes)
 Direct sequencing
cDNA chips
 Probes are cDNA fragments, usually amplified
by PCR
 Probes are deposited on a solid support,
either positively charged nylon or glass slide
 Samples (normally poly(A)+ RNA) are labelled
using fluorescent dyes
 At least two samples are hybridized to chip
 Fluorescence at different wavelengths
measured by a scanner
Standard protocol for
comparative hybridization

From Jeremy Buhler’s

Web pages
cDNA chip design
 Probe selection
 Non-redundant set of probes
 Includes genes of interest to project
 Corresponds to physically available clones
 Chip layout
 Grouping of probes by function
 Correspondance between wells in microtitre
plates and spots on the chip
Probe selection
 Make sure that database entries are cDNA
 Preference for RefSeq entries
 Criteria for non-redundancy
 >98% identity over >100 nt
 Accession number is unique
 Mapping of sequence to clone
 Use Unigene clusters
 Directly use data from sequence verified collection
(e.g. Research Genetics)
 Independently verify sequence
cDNA arrays on nylon and glass
 Nylon arrays
 Up to about 1000 probes per filter
 Use radiolabeled cDNA target
 Can use phosphorimager or X-ray film
 Glass arrays
 Up to about 40’000 probes per slide, or 10’000
per 2cm2 area (limited by arrayer’s capabilities)
 Use fluorescent targets
 Require specialized scanner
Glass chip manufacturing
 Choice of coupling method
 Physical (charge), non-specific chemical, specific
chemical (modified PCR primer)
 Choice of printing method
 Mechanical pins: flat tip, split tip, pin & ring
 Piezoelectric deposition (“ink-jet”)
 Robot design
 Precision of movement in 3 axes
 Speed and throughput
 Number of pins, numbers of spots per pin load
Typical Ink Jet Spot Deposition
Results

Volume per spot: 250 nl Volume per spot: 0.5 nl

Spot size: 1100 µm Spot size: 115
µm
Spot density: 70/cm2
Spot density:
Labelled BSA 4800/cm2
Typical Pin Spot Deposition
Microarray Results

(a.u.)
1 .5

t.
1.0

rel. fluor. in
0.5

7x11 microarray consisting of

identical Cy5-BSA spots (pitch 500
m)
Typical CV:  5%
Labelling and hybridization
 Targets are normally prepared by oligo(dT)
primed cDNA synthesis
 Probes should contain 3’ end of mRNA
 Need CoT1 DNA as competitor
 Specific activity will limit sensitivity of assay
 Alternative protocol is to make ds cDNA
containing bacterial promoter, then cRNA
 Can work with smaller amount of RNA
 Less quantitative
 Hybridization usually under coverslips
Scanning the arrays
 Laser scanners
 Excellent spatial resolution
 Good sensitivity, but can bleach fluorochromes
 Still rather slow
 CCD scanners
 Spatial resolution can be a problem
 Sensitivity easily adjustable (exposure time)
 Faster and cheaper than lasers
 In all cases, raw data are images showing
fluorescence on surface of chip
Zeptosens : Planar Waveguide Principle - for High Sensitivity
Fluorescence Microarray Detection

free label

microarray
on chip
excitation of bound label

Imaging
of surface-confined fluorescence

CCD camera
The Affymetrix approach
 Probes are oligos synthesized in situ using a
photolithographic approach
 There are at least 5 oligos per cDNA, plus an
equal number of negative controls
 The apparatus requires a fluidics station for
hybridization and a special scanner
 Only a single fluorochrome is used per
hybridization
 It is very expensive !
Affymetrix chip production

Video Clip
Commercial chips
 Clontech, Incyte, Research Genetics -
filter-based arrays with up to about 8000
clones
 Incyte / Synteni - 10’000 probe chips, not
distributed (have to send them target
RNA)
 Affymetrix - oligo-based chips with 12’000
genes of known function (16 oligos/gene)
and 4x10’000 genes from ESTs
Alternative technologies
 Synthesis of probes on microbeads
 Hybridization in solution
 Identification of beads by fluorescent bar
coding by embedding transponders
 Readout using micro-flow cells or optic fiber
arrays
 Production of “universal” arrays
 Array uses a unique combination of oligos, and
probes containing the proper complements
Fiber optics technology

 
                                                                                                                   
                                                                                                                 

To learn more: Illumina’s Web site

Arrays for genetic analysis
 Mutation detection
 Oligos (Affymetrix type) representing all known
alleles
 PCR followed by primer extension, with
detection of alleles by MALDI-TOF mass
spectroscopy (Sequenom)
 Gene loss and amplification
 Measure gene dosage in genomic DNA by
hybridization to genomic probes
Microarray data on the Web
 Many groups have made their raw data available,
but in many formats
 Some groups have created searchable databases
 There are several initiatives to create “unified”
databases
 EBI: ArrayExpress
 NCBI: Gene Expression Omnibus
 Companies are beginning to sell microarray
expression data (e.g. Incyte)
Bioinformatics of microarrays
 Array design: choice of sequences to be used as
probes
 Analysis of scanned images
 Spot detection, normalization, quantitation
 Primary analysis of hybridization data
 Basic statistics, reproducibility, data scattering, etc.
 Comparison of multiple samples
 Clustering, SOMs, classification …
 Sample tracking and databasing of results
Web links
 Leming Shi’s Gene-Chips.com page – very
rich source of basic information and
commercial and academic links
 DNA chips for dummies animation
 A step by step description of a microarray
experiment by Jeremy Buhler
 The Big Leagues: Pat Brown and NHGRI
microarray projets