Genomic Arrays – an overviewGenomic Arrays – an overview

Dr. Colin CampbellDr. Colin Campbell

Transcription

Genome

AAAAA

Regulation

DNA mRNA Protein

The Central Dogma

TranscriptionTranscription TranslationTranslation

Genomics in perspectiveGenomics in perspective

Sequences available for hundreds of genomesSequences available for hundreds of genomesviruses/plasmids >> mammalian genomesviruses/plasmids >> mammalian genomes

Genome sequence only the startGenome sequence only the start

Need to understand:Need to understand:

genomic structure, replication, expression genomic structure, replication, expression

Problem of Problem of scale, complexity and diversityscale, complexity and diversity

Advent of HTS functional genomic technologies:Advent of HTS functional genomic technologies:

microarraymicroarray, Si RNA, mutagenesis, proteomics, imaging, Si RNA, mutagenesis, proteomics, imaging

Post Genomic ChallengesPost Genomic Challenges

Functional genomics toolboxFunctional genomics toolbox

all genes used to assemble an organismall genes used to assemble an organism

ClassifyClassify IdentifyIdentifySequenceSequence ascribe functionascribe function Monitor expressionMonitor expression

Post genomic approachesPost genomic approaches

Microarrays – a post genomic technologyMicroarrays – a post genomic technology

Gene Expression/GenotypingGene Expression/Genotyping

ProteomicsProteomics

Fundamental and applied biomedical researchFundamental and applied biomedical research

Supporting TechnologiesSupporting Technologies

Statistics/BioinformaticsStatistics/Bioinformatics

HTS Technology Developments: HTS Technology Developments: ArrayingArraying/ Scanning/ Lab-on-a-chip/ Scanning/ Lab-on-a-chip

Computing/ DatabasesComputing/ Databases

Mammalian GenomeMammalian Genome DatabaseDatabase

Traditional method:Traditional method:taking gene by gene approachtaking gene by gene approach

InsufficientInsufficient to meet magnitude of problemto meet magnitude of problem

Array technologyArray technology

Developed to provide a systematic way of studying RNA expression, Developed to provide a systematic way of studying RNA expression, genotyping, DNA/ RNA interactions and numerous other applicationsgenotyping, DNA/ RNA interactions and numerous other applications

Array = A regular or uniform arrangementArray = A regular or uniform arrangement

e.g. of DNA probes or other elements such as proteins ore.g. of DNA probes or other elements such as proteins ortissue sections arranged on glass slides or nylon membranestissue sections arranged on glass slides or nylon membranes

Evolution of array technologyEvolution of array technology

Transcription

Genome

AAAAA

Regulation

DNA mRNA Protein

The Central Dogma

TranscriptionTranscription TranslationTranslation

RNA transcription analysisRNA transcription analysisExpression of RNA assessed by Northern blotting, RNAase protection, Expression of RNA assessed by Northern blotting, RNAase protection, RT-PCR methodsRT-PCR methods

Low to medium throughput approaches.Low to medium throughput approaches.Do not easily accommodate scale, complexity and diversity challengesDo not easily accommodate scale, complexity and diversity challenges

e.g. Northern Blote.g. Northern Blot

DNADNA

mRNAmRNA

proteinsproteins

DenatureDenature

Gel electrophoresis,Gel electrophoresis,RNA separated by RNA separated by

Size and blottedSize and blottedon filteron filter

Filters exposed to labelledFilters exposed to labelled DNA probe and subjectDNA probe and subject

to radiographyto radiography

RNA transcripts anlysed singly. RNA transcripts anlysed singly. Definiton of transcriptome would Definiton of transcriptome would take thousands of blots take thousands of blots

CellCell

DNADNAGENOMEGENOME

cDNA(s) or oligonucleotide(s)cDNA(s) or oligonucleotide(s) representative of genesrepresentative of genes

spotted on slidespotted on slide

11

22

33

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genesgenes

DNADNA

mRNAmRNA

proteinsproteins

ReverseReversetranscribe RNAtranscribe RNA

Using Using Cy3 (test RNA)Cy3 (test RNA) oror Cy5 (control) Cy5 (control) dCTPdCTP

Test cDNATest cDNA

control cDNAcontrol cDNA

IntensityIntensityvaluevalue

Intensity Intensity valuevalue

Hybridise to arrayHybridise to array

==RelativeRelativeValueValue

+ve = upreg+ve = upregArray

Relative expression of RNARelative expression of RNAdefined at whole genome leveldefined at whole genome level

The microarray solutionThe microarray solution

First attempts at exploiting array approachesFirst attempts at exploiting array approaches involved filter based screening of clone libraries involved filter based screening of clone libraries

Basic genomic and RNA expression studiesBasic genomic and RNA expression studies

Two key innovations have enhanced the utility of genomic microarraysTwo key innovations have enhanced the utility of genomic microarrays

1. Use of glass substrates to construct miniaturised arrays1. Use of glass substrates to construct miniaturised arraysDIRECT DEPOSITION: Using automated printers: ~30-40K DNA probe elements DIRECT DEPOSITION: Using automated printers: ~30-40K DNA probe elements deposited on a glass slidedeposited on a glass slideIN SITU SYNTHESIS: several million individual DNA probe elements IN SITU SYNTHESIS: several million individual DNA probe elements defined by photolithography on silicon wafersdefined by photolithography on silicon wafers

2. The use of fluorescence for detection2. The use of fluorescence for detection

Microarray optionsMicroarray options

Method 1. Array of 5,000 mouse genesMethod 1. Array of 5,000 mouse genes - direct deposition method - direct deposition method

DNADNAGENOMEGENOME

cDNA(s) or oligonucleotide(s)cDNA(s) or oligonucleotide(s) representative of genesrepresentative of genes

spotted on slidespotted on slide

11

22

33

44

11

22

33

44

genesgenes

DNADNA

mRNAmRNA

proteinsproteins

ReverseReversetranscribe RNAtranscribe RNA

Using Using Cy3 (test RNA)Cy3 (test RNA) oror Cy5 (control) Cy5 (control) dCTPdCTP

Test cDNATest cDNA

control cDNAcontrol cDNA

IntensityIntensityvaluevalue

Intensity Intensity valuevalue

Hybridise to arrayHybridise to array

==RelativeRelativeValueValue

+ve = upreg+ve = upregArray

Relative expression of RNARelative expression of RNAdefined at whole genome leveldefined at whole genome level

The microarray solutionThe microarray solution

Direct deposition DNA microarray scanner imageDirect deposition DNA microarray scanner image

LL TT

Method 2. In situ synthesised oligo array - Affymetrix GeneChipMethod 2. In situ synthesised oligo array - Affymetrix GeneChip® system® system

GG AAAAAA GGGGGG CCCCCC TTTTTT AA GG CCTT AA GG CCTT AA GGTT

3’3’

Gene SequenceGene Sequence

25 mer25 mer

Many million fold bound in specific featureMany million fold bound in specific feature

representative DNA sequences derived from 3’ end of generepresentative DNA sequences derived from 3’ end of gene

20 features used to represent one gene20 features used to represent one gene

400,000 features per 400,000 features per array representing array representing

~ 12,000 genes~ 12,000 genes

DNADNA

Cell/ Tissue of interestCell/ Tissue of interest

AAAAAA

AAAAAAAAAAAA

AAAAAA Isolation of Isolation of total RNAtotal RNA AAAAAA

AAAAAA

AAAAAA

AAAAAA

TTTTTT

TTTTTT

TTTTTT

TTTTTT

1st strand cDNA synthesis1st strand cDNA synthesis

T7 Promoter incorporatedT7 Promoter incorporated in first strand synthesisin first strand synthesis

TTTTTT

TTTTTT

TTTTTT

TTTTTT

2nd strand cDNA synthesis2nd strand cDNA synthesis

ds cDNAds cDNA

Affymetrix target labellingAffymetrix target labelling

bb

bb

bb

bb

TTTTTT

TTTTTT

TTTTTT

TTTTTT

In vitro In vitro transcription usingtranscription usingBiotinylated dNTPsBiotinylated dNTPs

Affymetrix labelling and hybridisationAffymetrix labelling and hybridisation

Biotinylated cRNABiotinylated cRNA

bb

bb

bb

bb LLLL

LLLL

bb

bb

bb

bb

Hybridise to ArrayHybridise to Array

SASA

SASA

SASA

SASA

Affymetrix Gene Chip resultsAffymetrix Gene Chip results

Expression of 10K genes – but what is the result ?Expression of 10K genes – but what is the result ?

Statistics and Bioinformatics essentialStatistics and Bioinformatics essential

advantagesadvantages

Scale - true global analyses possibleScale - true global analyses possible

Semi-quantitativeSemi-quantitative

High throughputHigh throughput

Emerging standards for analysisEmerging standards for analysis

Scale demands stringent QC and analytical routinesScale demands stringent QC and analytical routines

disadvantagesdisadvantages

SensitivitySensitivity

PrecisionPrecision

Relative cost/logisticsRelative cost/logistics

Context independentContext independent

Microarray technology - pros and consMicroarray technology - pros and cons

Microarrays in cancer biologyMicroarrays in cancer biology

RNA Expression profiling arrays: Targets > pathwaysRNA Expression profiling arrays: Targets > pathwaysGenotyping arrays: HTS SNP analysis > gene association studiesGenotyping arrays: HTS SNP analysis > gene association studiesProtein arrays: marker setsProtein arrays: marker sets

Expression based classification to detect dominant patterns of Expression based classification to detect dominant patterns of expression in heterogeneous tumoursexpression in heterogeneous tumours

Can identify:Can identify:

•Tumour markersTumour markers•Origin of tumourOrigin of tumour•Developmental stageDevelopmental stage•Metastatic potential Metastatic potential •Therapeutic response profileTherapeutic response profile•Fundamental insights >> definition of cancer pathways and controlFundamental insights >> definition of cancer pathways and control•Contribute to diagnosis, prognosis and therapy.Contribute to diagnosis, prognosis and therapy.

Interferon relatedInterferon related

Clustered gene setsClustered gene sets

Breast luminal cell profileBreast luminal cell profile

Basal epithelial cell profileBasal epithelial cell profile

Lung adenocarcinomaLung adenocarcinoma enriched profileenriched profile

Proliferation gene setProliferation gene set

DNA microarrays now extensively employed for DNA microarrays now extensively employed for RNA expression profilingRNA expression profilingstudies in biomedical research. studies in biomedical research.

Crucial role for statistics, bioinfomatics and computational science to turn HTS data into Crucial role for statistics, bioinfomatics and computational science to turn HTS data into useful information (gene targets and pathway definition) for the biologist to interpretuseful information (gene targets and pathway definition) for the biologist to interpret

Provides a critical approach to a thorough understanding of fundamental biological Provides a critical approach to a thorough understanding of fundamental biological processes. Also contributing to applied areas such as disease diagnosis and definition.processes. Also contributing to applied areas such as disease diagnosis and definition. DNA microarrays providing a HTS and global platform technology for numerous DNA microarrays providing a HTS and global platform technology for numerous biomedical and genomic research applicationsbiomedical and genomic research applications

- splicing- splicing- sequencing and SNP analysis (v. high density oligo arrays under development)- sequencing and SNP analysis (v. high density oligo arrays under development)- CGH, BAC clones- CGH, BAC clones- epigenetic studies e.g. DNA methylation- epigenetic studies e.g. DNA methylation- Also, platforms developing for: proteins, cells and tissues- Also, platforms developing for: proteins, cells and tissues

DNA microarray approaches will ultimately replace many of the standard methods genetic DNA microarray approaches will ultimately replace many of the standard methods genetic

analysis.analysis.

DNA microarrays – a platform technologyDNA microarrays – a platform technology

Full definition of biological processes requires additional contextualFull definition of biological processes requires additional contextualinforrmation (e.g. spatial, temporal, modification) inforrmation (e.g. spatial, temporal, modification)

Methods for precise micro sampling of complex cell populations and tissuesMethods for precise micro sampling of complex cell populations and tissuescan be combined with microarray readouts.can be combined with microarray readouts.

Initial step involves precise sampling via cell sorting/enrichment or micro-dissectionInitial step involves precise sampling via cell sorting/enrichment or micro-dissectiontechniquestechniques

Combine with target sample (micro RNA sample) amplification methods to enableCombine with target sample (micro RNA sample) amplification methods to enable readout on standard DNA microarray platformsreadout on standard DNA microarray platforms

Increases power of analysis and biological interpretationIncreases power of analysis and biological interpretation

Biological contextBiological context

Array technology will continue to develop for DNA, RNA, protein and various Array technology will continue to develop for DNA, RNA, protein and various other physiological measurements. other physiological measurements.

Developments will require increasing interface of biology with physical sciences Developments will require increasing interface of biology with physical sciences and technology.and technology.

Allow new questions to be asked at the whole genome/proteome level.Allow new questions to be asked at the whole genome/proteome level.

Integration of HTS genomic, proteomic and cellular readouts will be required to Integration of HTS genomic, proteomic and cellular readouts will be required to define biological complexity and approach systems level understandingdefine biological complexity and approach systems level understanding

Key to this is input from bioinformatics and computational science to analyse, Key to this is input from bioinformatics and computational science to analyse, store and visualise datastore and visualise data

Future potential in biology and medicineFuture potential in biology and medicine