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
44
11
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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