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Genomic Arrays – an overview. Dr. Colin Campbell. The Central Dogma. Genome. Regulation. AAAAA. Transcription. Transcription. Translation. Protein. DNA. mRNA. Genomics in perspective. Post Genomic Challenges.

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Presentation Transcript
slide2

The Central Dogma

Genome

Regulation

AAAAA

Transcription

Transcription

Translation

Protein

DNA

mRNA

slide4

Post Genomic Challenges

Sequences available for hundreds of genomesviruses/plasmids >> mammalian genomesGenome sequence only the startNeed to understand:genomic structure, replication, expression Problem of scale, complexity and diversityAdvent of HTS functional genomic technologies:microarray, Si RNA, mutagenesis, proteomics, imaging

post genomic approaches
Post genomic approaches

Functional genomics toolbox

ascribe function

Monitor expression

Sequence

Classify

Identify

all genes used to assemble an organism

slide6

Microarrays – a post genomic technology

Mammalian Genome

Database

Gene Expression/Genotyping

Proteomics

Fundamental and applied biomedical research

Supporting Technologies

Statistics/Bioinformatics

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

Computing/ Databases

evolution of array technology
Evolution of array technology

Traditional method:

taking gene by gene approach

Insufficientto meet magnitude of problem

Array technology

Developed to provide a systematic way of studying RNA expression,

genotyping, DNA/ RNA interactions and numerous other applications

Array = A regular or uniform arrangement

e.g. of DNA probes or other elements such as proteins or

tissue sections arranged on glass slides or nylon membranes

slide8

The Central Dogma

Genome

Regulation

AAAAA

Transcription

Transcription

Translation

Protein

DNA

mRNA

slide9

RNA transcription analysis

Expression of RNA assessed by Northern blotting, RNAase protection, RT-PCR methods

Low to medium throughput approaches.

Do not easily accommodate scale, complexity and diversity challenges

e.g. Northern Blot

Filters exposed to labelled

DNA probe and subject

to radiography

Cell

DNA

Gel electrophoresis,

RNA separated by

Size and blotted

on filter

mRNA

Denature

proteins

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

the microarray solution
The microarray solution

cDNA(s) or oligonucleotide(s)

representative of genes

spotted on slide

Intensity

value

1

Relative

Value

=

Intensity

value

2

1

+ve = upreg

Array

2

3

genes

3

4

4

DNA

GENOME

Hybridise to array

Test cDNA

control cDNA

DNA

Reverse

transcribe RNA

Using Cy3 (test RNA)

orCy5 (control) dCTP

Relative expression of RNA

defined at whole genome level

mRNA

proteins

microarray options
Microarray options

First attempts at exploiting array approaches

involved filter based screening of clone libraries

Basic genomic and RNA expression studies

Two key innovations have enhanced the utility of genomic microarrays

1. Use of glass substrates to construct miniaturised arrays

DIRECT DEPOSITION: Using automated printers: ~30-40K DNA probe elements

deposited on a glass slide

IN SITU SYNTHESIS: several million individual DNA probe elements

defined by photolithography on silicon wafers

2. The use of fluorescence for detection

the microarray solution13
The microarray solution

cDNA(s) or oligonucleotide(s)

representative of genes

spotted on slide

Intensity

value

1

Relative

Value

=

Intensity

value

2

1

+ve = upreg

Array

2

3

genes

3

4

4

DNA

GENOME

Hybridise to array

Test cDNA

control cDNA

DNA

Reverse

transcribe RNA

Using Cy3 (test RNA)

orCy5 (control) dCTP

Relative expression of RNA

defined at whole genome level

mRNA

proteins

slide15

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

G

G

G

G

G

G

G

T

T

T

T

T

T

A

A

A

A

A

A

C

C

C

C

C

Gene Sequence

representative DNA sequences derived from 3’ end of gene

3’

25 mer

T

L

Many million fold bound in specific feature

20 features used to represent one gene

400,000 features per array representing

~ 12,000 genes

affymetrix target labelling

TTT

TTT

TTT

TTT

AAA

AAA

AAA

AAA

AAA

AAA

AAA

Affymetrix target labelling

Cell/ Tissue of interest

2nd strand cDNA synthesis

1st strand cDNA synthesis

DNA

TTT

TTT

Isolation of

total RNA

ds cDNA

AAA

TTT

TTT

T7 Promoter incorporated

in first strand synthesis

slide17

b

b

b

b

b

SA

SA

SA

SA

Affymetrix labelling and hybridisation

In vitro transcription using

Biotinylated dNTPs

Hybridise to Array

Biotinylated cRNA

TTT

L

b

TTT

L

b

b

TTT

b

L

b

TTT

b

L

b

slide18

Affymetrix Gene Chip results

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

Statistics and Bioinformatics essential

microarray technology pros and cons
Microarray technology - pros and cons

Scale - true global analyses possible

Semi-quantitative

advantages

High throughput

Sensitivity

Precision

Scale demands stringent QC and analytical routines

Emerging standards for analysis

disadvantages

Relative cost/logistics

Context independent

microarrays in cancer biology
Microarrays in cancer biology
  • RNA Expression profiling arrays: Targets > pathways
  • Genotyping arrays: HTS SNP analysis > gene association studies
  • Protein arrays: marker sets
  • Expression based classification to detect dominant patterns of expression in heterogeneous tumours
  • Can identify:
  • Tumour markers
  • Origin of tumour
  • Developmental stage
  • Metastatic potential
  • Therapeutic response profile
  • Fundamental insights >> definition of cancer pathways and control
  • Contribute to diagnosis, prognosis and therapy.
slide21

Clustered gene sets

Interferon related

Breast luminal cell profile

Basal epithelial cell profile

Lung adenocarcinoma

enriched profile

Proliferation gene set

slide22

DNA microarrays – a platform technology

DNA microarrays now extensively employed for RNA expression profilingstudies in biomedical research. Crucial role for statistics, bioinfomatics and computational science to turn HTS data into useful information (gene targets and pathway definition) for the biologist to interpretProvides a critical approach to a thorough understanding of fundamental biological processes. Also contributing to applied areas such as disease diagnosis and definition.DNA microarrays providing a HTS and global platform technology for numerous biomedical and genomic research applications- splicing- sequencing and SNP analysis (v. high density oligo arrays under development)- CGH, BAC clones- epigenetic studies e.g. DNA methylation- Also, platforms developing for: proteins, cells and tissuesDNA microarray approaches will ultimately replace many of the standard methods genetic analysis.

slide23

Biological context

Full definition of biological processes requires additional contextual

inforrmation (e.g. spatial, temporal, modification)

Methods for precise micro sampling of complex cell populations and tissues

can be combined with microarray readouts.

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

techniques

Combine with target sample (micro RNA sample) amplification methods to enable

readout on standard DNA microarray platforms

Increases power of analysis and biological interpretation

slide24

Future potential in biology and medicine

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

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

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

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

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