Biological background: Molecular Laboratory Techniques

Class web site:

http://statwww.epfl.ch/davison/teaching/Microarrays/ETHZ/

Statistics for Microarrays

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

Reverse transcriptase makes a complementary DNA copy from RNA.

A virus is a nucleic acid in a protein coat.

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

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)

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

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

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

Ngai Lab arrayer , UC Berkeley

Building the chip

Print-tip head

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

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 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)