1. Statistics for Microarrays
Biological background: Molecular Laboratory Techniques
Class web site:

2. Molecular Laboratory Techniques
Hybridizing DNA
Copying DNA
Cutting DNA
Probing DNA

3. 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

5. 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

6. 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

8. (RT)

9. Types of Viruses A virus is a nucleic acid in a protein coat.
Reverse transcriptase makes a complementary DNA copy from RNA.

10. Reverse transcription
Clone cDNA strands, complementary to the mRNA
mRNA
G U A A U C C U C
Reverse transcriptase
cDNA
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
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

11. RT-PCR

12. Restriction Enzymes Cut DNA

13. 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)

14. 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.

15. 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

16. 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

17. 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

18. Pins collect cDNA from wells
384 well plate --
Contains cDNA probes
cDNA clones
Spotted in duplicate
Print-tip group 1
Glass Slide
Array of bound cDNA probes
4x4 blocks = 16 print-tip groups
Print-tip group 6

19. Building the chip
Ngai Lab arrayer , UC Berkeley
Print-tip head

20. cDNA microarrays Compare gene expression in two samples PRINT SAMPLES
cDNA from one gene on each spot
SAMPLES
cDNA labelled red/green
e.g. treatment / control
or normal / tumor tissue

21. HYBRIDIZE
Add equal amounts of labelled cDNA samples to microarray.
SCAN
Laser
Detector

22. Yeast genome on a chip

23. Web animation of a cDNA microarray experiment

24. 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

25. 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

26. 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

27. Scanning the arrays Laser scanners CCD 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

28. 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

29. Affymetrix chip production

30. 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

31. 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

32. 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) 4

33. 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

34. 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)

35. 16-bit TIFF files (Rfg, Rbg), (Gfg, Gbg) R, G 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
Discrimination
Biological verification
and interpretation