DNA chips

1
DNA chips

• Technologies and utility

2
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

3
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

4
Major technologies

• cDNA probes (gt 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

5
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

6
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

7
Standard protocol for comparative hybridization
From Jeremy BuhlersWeb pages
8
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

9
Probe selection

• Make sure that database entries are cDNA
• Preference for RefSeq entries
• Criteria for non-redundancy
• gt98 identity over gt100 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

10
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 40000 probes per slide, or 10000
  per 2cm2 area (limited by arrayers capabilities)
• Use fluorescent targets
• Require specialized scanner

11
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

12
Typical Ink Jet Spot Deposition Results
Volume per spot 0.5 nl Spot size
115 µm Spot density 4800/cm2
Volume per spot 250 nl Spot size
1100 µm Spot density 70/cm2
Labelled BSA
13
Typical Pin Spot Deposition Microarray Results
rel. fluor. int. (a.u.)
7x11 microarray consisting of identical Cy5-BSA
spots (pitch 500 mm)
Typical CV ? 5
14
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

15
Scanning the arrays

• Laser scanners
• Excellent spatial resolution
• Good sensitivity, but can bleach fluorochromes
• Still rather slow
• New technology (Agilent) auto-focus of laser
  beam on chip surface
• 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

16
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
17
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 !

18
Affymetrix chip production
19
Properties of Affy chips

• Major advantages
• Very robust protocols and high reproducibility
• Widely used, so annotation of probe sets is of
  relatively high quality
• Disadvantages
• Single target hybridization, so comparison always
  involves two experiments, and dye swaps are
  impossible
• Match/mismatch technology has major limitations
  mismatch signal often higher than match, and dose
  response curve is different for each pair

20
Commercial chips

• Clontech, Incyte, Invitrogen- filter-based arrays
  with up to about 8000 clones
• Agilent long oligo (60 nt) glass chips with
  20,000 probes for human, mouse, rat also sells
  cDNA based arrays
• Affymetrix - oligo-based chips containing
  typically 20,000 probe sets per chip, each with
  5-20 match/mismatch pairs

21
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
• Design similar to the tag/anti-tag system in
  Megacloning
• Tags remain the same, probes attached to
  anti-tags can be customized

22
Fiber optics technology
To learn more Illuminas Web site
23
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 (CGH arrays)

24
Other uses of microarrays

• Systematic survey of transcriptional activity in
  genome region and detection of exonic regions
• Comparative genomics, especially for closely
  related species
• These applications require chips covering entire
  genomes Perlegen, spin-off of Affymetrix

25
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)

26
The problem of data exchange

• Microarray experiments are by definition
  idiosyncratic
• Probes used, RNA source, labelling protocols,
  hybridization conditions, etc.
• Standards are needed to exchange information
• Information about experiments MIAME
• Data exchange format MAGE
• Ontology for description of target sources OWG
• Data transformation and normalization methods
  (required to compare expression levels)
• Coordination MGED Society

27
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

28
Web links

• Leming Shis 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