Biological background: Molecular Laboratory
Techniques
Class web site: http://statwww.epfl.ch/davison/teaching/Microarrays/ETHZ/

• 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

• 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

• 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)

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

Clone cDNA strands, complementary to the mRNA mRNA G U A A U C C U C
Reverse transcriptase cDNA
C A T T A G G A G
C A T T A G G A G

• 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)

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.

• 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

• 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

• 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

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

Ngai Lab arrayer , UC Berkeley Print-tip head

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.
Laser
SCAN
Detector

Yeast genome on a chip

http://www.bio.davidson.edu/courses/genomics/chip/ chip.html

• 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

• 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 2cm 2 area (limited by arrayer’s capabilities)
– Use fluorescent targets
– Require specialized scanner

• 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

• 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

• 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

• 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

• 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

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)

• 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

• 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)

Estimation
Biological question
Differentially expressed genes
Sample class prediction etc.
Experimental design
Microarray experiment
16-bit TIFF files
Image analysis
(Rfg, Rbg) , (Gfg, Gbg)
Testing
Normalization
R , G
Clustering Discrimination
Biological verification and interpretation