Transcript Document

Statistics for Microarrays
Biological background: Molecular Laboratory
Techniques
Class web site:
http://statwww.epfl.ch/davison/teaching/Microarrays/ETHZ/
Molecular Laboratory Techniques
• Hybridizing DNA
• Copying DNA
• Cutting DNA
• Probing DNA
Hybridization
• Hybridization exploits a potent feature
of the DNA duplex – the sequence
complementarity of the two strands
• Strands can be separated (denatured)
by heating
• Remarkably, DNA can reassemble with
perfect fidelity from separated
strands
Polymerase Chain Reaction (PCR)
• PCR is used to amplify (copy) specific DNA
sequences in a complex mixture when the ends of
the sequence are known
• Source DNA is denatured into single strands
• Two synthetic oligonucleotides complementary to
the 3’ ends of the segment of interest are added
in great excess to the denatured DNA, then the
temperature is lowered
• The genomic DNA remains denatured since the
complementary strands are at too low a
concentration to encounter each other during the
period of incubation
• The specific oligonucleotides hybridize with
complementary sequences in the genomic DNA
PCR, ctd
• The hybridized oligos then serve as primers for
DNA synthesis, which begins upon addition of a
supply of nucleotides and a temperature
resistant polymerase such as Taq polymerase,
from Thermus aquaticus (a bacterium that lives
in hot springs)
• Taq polymerase extends the primers at
temperatures up to 72˚C
• When synthesis is complete, the whole mixture
is heated further (to 95˚C) to melt the newly
formed duplexes
• Repeated cycles (25—30) of synthesis (cooling)
and melting (heating) quickly provide many DNA
copies
(RT)
Types of Viruses
A virus is a nucleic acid in a protein coat.
Reverse transcriptase makes a complementary
DNA copy from RNA.
Reverse transcription
Clone cDNA strands, complementary to the mRNA
mRNA
G U AA U C C U C
Reverse
transcriptase
cDNA
CATTAG GAG
CA
G
CT
ACTTAA
TTG
ATG
GAA
G
AGGA G
G
C
A
T
T
A
G
C A T T A G G AGGA G
RT-PCR
Restriction Enzymes Cut DNA
Probing DNA
• One way to study a specific DNA fragment within
a genome is to probe for the sequence of the
fragment
• A probe is a labeled (usually radioactive or
fluorescent) single-stranded oligonucleotide,
synthesized to be complementary to the sequence
of interest – probe sequence is known
• Attach single-stranded DNA to a membrane (or
other solid support) and incubate with the probe
so that it hybridizes
• Visualize the probe (e.g. by X-ray for radioactive
probes)
Measuring Gene Expression
Idea: measure the amount of mRNA to see which
genes are being expressed in (used by) the cell.
Measuring protein might be more direct, but is
currently harder.
Principal Uses of Microarrays
• Genome-scale gene expression analysis
– Differential gene expression between two (or
more) sample types
– Responses to environmental factors
– Disease processes (e.g. cancer)
– Effects of drugs
– Identification of genes associated with clinical
outcomes (e.g. survival)
• Detection of sequence variation
– Genetic typing
– Detection of somatic mutations (e.g. in oncogenes)
– Direct sequencing
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
Brief outline of steps for
producing a cDNA microarray
• 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
Pins collect
cDNA from
wells
384 well
plate -Print-tip
group 1
cDNA clones
Spotted in duplicate
Contains cDNA
probes
Glass Slide
Array of bound cDNA
probes
4x4 blocks = 16 print-tip
groups
Print-tip
group 6
Building the chip
Ngai Lab arrayer , UC Berkeley
Print-tip head
cDNA microarrays
Compare gene expression in two samples
PRINT
cDNA from one
gene on each spot
SAMPLES
cDNA labelled red/green
e.g. treatment / control
or
normal / tumor tissue
HYBRIDIZE
Add equal amounts of
labelled cDNA samples to
microarray.
SCAN
Laser
Detector
Yeast genome on a chip
Web animation of a cDNA
microarray experiment
http://www.bio.davidson.edu/courses/genomics/chip/
chip.html
cDNA Microarray 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
– Correspondence between wells in
microtiter plates and spots on the chip
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
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
Affymetrix GeneChips
• 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
• Expensive, but getting cheaper
Affymetrix chip production
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
cDNA microarray experiments
mRNA levels compared in many different
contexts
• Different tissues, same organism (brain v.
liver)
• Same tissue, same organism (ttt v. ctl,
tumor v. non-tumor)
• Same tissue, different organisms (wt v. ko,
tg, or mutant)
• Time course experiments (effect of ttt,
development)
• Other special designs (e.g. to detect spatial
patterns)
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
• Several initiatives to create “unified” databases
– EBI: ArrayExpress
– NCBI: Gene Expression Omnibus
• Some companies are beginning to sell microarray
expression data (e.g. Incyte)
Biological question
Differentially expressed genes
Sample class prediction etc.
Experimental design
Microarray experiment
16-bit TIFF files
Image analysis
(Rfg, Rbg), (Gfg, Gbg)
Normalization
R, G
Estimation
Testing
Clustering
Biological verification
and interpretation
Discrimination