biological background: molecular laboratory techniques class web site: statistics for microarrays
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Biological background: Molecular Laboratory Techniques
Class web site:
http://statwww.epfl.ch/davison/teaching/Microarrays/ETHZ/
Statistics for Microarrays
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Molecular Laboratory Techniques
• Hybridizing DNA
• Copying DNA
• Cutting DNA
• Probing DNA
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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
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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
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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
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(RT)
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Types of Viruses
Reverse transcriptase makes a complementary DNA copy from RNA.
A virus is a nucleic acid in a protein coat.
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Reverse transcription
Clone cDNA strands, complementary to the mRNA
G U A A U C C U C
Reverse transcriptase
mRNA
cDNA
C A T T A G G A G C A T T A G G A G C A T T A G G A G C A T T A G G A G
T T A G G A G
C A T T A G G A G C A T T A G G A G C A T T A G G A G
C A T T A G G A G
C A T T A G G A G
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RT-PCR
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Restriction Enzymes Cut DNA
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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)
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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.
Measuring Gene Expression
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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
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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
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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
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384 well plate --
Contains cDNA probes
Glass SlideArray of bound cDNA probes
4x4 blocks = 16 print-tip groups
Print-tip group 6
cDNA clones
Spotted in duplicate
Print-tip group 1
Pins collect cDNA from wells
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Ngai Lab arrayer , UC Berkeley
Building the chip
Print-tip head
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cDNA microarraysCompare gene expression in two samples
PRINTcDNA from one gene on each spot
SAMPLEScDNA labelled red/green
e.g. treatment / control
or normal / tumor tissue
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HYBRIDIZE
Add equal amounts of labelled cDNA samples to microarray.
SCAN
Laser Detector
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Yeast genome on a chip
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Web animation of a cDNA microarray experiment
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
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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
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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
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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
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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
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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
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Affymetrix chip production
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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
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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
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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)
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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
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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)
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Biological questionDifferentially expressed genesSample class prediction etc.
Testing
Biological verification and interpretation
Microarray experiment
Estimation
Experimental design
Image analysis
Normalization
Clustering Discrimination
R, G
16-bit TIFF files
(Rfg, Rbg), (Gfg, Gbg)