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Lecture 5: Challenges in the post-
genomic era
The tiger leg leaf frogPhoto: Zig LeszccynskiImage: courtesy Rainforest Alliance
Are genome projects the end?
Functional Genomics Projects Genomics & Genetics Resources
Human Genome Project:
To access the Human Genome Project (HGP) research programme resources here at the WTSI
Copy Number Variation:
The CNV project examines copy number changes in the population
Cancer Genome Project:
Identifying somatically acquired sequence variants/mutations and hence genes critical in the development of human cancers
Genomic Microarrays:
Detailed analysis of genomic copy number changes in tumours
Immunogenomics:
Genetics and epigenetics of the immune subgenome and their roles in evolution and disease
Microarray Facility:
Development and use of high-density arrays for expression profiling
Molecular Cytogenetics:
Chromosome organisation and structure in human disease
Molecular Genetics & Proteomics:
Uses of human chromosome 22 as a model system for genomic
Molecular Cytogenetics:
Chromosome organisation and structure in human disease
Molecular Genetics & Proteomics:
Uses of human chromosome 22 as a model system for genomic analysis
Mouse Genomics:
Investigations of the function of genes using the mouse as model genetic system
Genetrap:
Sanger Institute Gene Trap Resource
Fission Yeast Functional Genomics:
Research focuses on genome-wide gene expression profiles and regulatory networks using DNA microarrays and molecular genetics
Atlas of Gene Expression:
Describing different cell types in adult/embryonic tissues alongside a description of expression level/localisation of protein products
Human Epigenome Project (HEP):
Provides an epigenetic resource of genome-wide DNA methylation reference profiles in human tissues and cell lines.
Malaria:
Understanding malaria from basic biology to practical applications
Pathogen Arrays:
Comparative genomics and expression profiling of small genomes
MICER:
Mutagenic Insertion and Chromosome Engineering Resource
ENCODE:
Encyclopedia Of DNA Elements project sets out to identify all functional elements in the human genome sequence
DECIPHER:
DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources
http://www.sanger.ac.uk/PostGenomics/
Functional genomics(A) Identifying genes from the sequence (B) Gene expression profiling
(transcriptomics)(C) Model systems
Proteomics
Systems biology
Post-genomics
(A) Hunting genes from the sequence
2 broad approaches1) Ab initio method (computational)
Codon analysis Regulatory regions (TF binding sites, CpG
islands etc) Exon-intron boundaries
2) Experimental method Hybridisation approaches – Northern
Blots, cDNA capture / cDNA select, Zoo blots
Transcript mapping: RT-PCR, exon trapping etc
Northern Blot Zoo Blot
Transcriptomecomplete collection of transcribed
elements of the genome (global mRNA profiling)
transcriptome maps provide clues on • Regions of transcription• Transcription factor binding sites • Sites of chromatin modification • Sites of DNA methylation • Chromosomal origins of replication
(B) Gene expression profiling
The transcriptome
Advantages: high-throughput information Gene expression profile of the
cell/tissue
problems false –positives data analysisCost
Analysed by DNA Microarrays
Microarrays….
gene inactivation methods (knockouts, RNAi, site-directed mutagenesis, transposon tagging, genetic footprinting etc)
Gene overexpression methods (knock-ins, transgenics, reporter genes)
(C) MODEL SYSTEMS
RNAi
RNAi mimics loss-of-function mutations
Non-inheritable
Lack of reproducibility
How does RNAi work?
http://www.nature.com/focus/rnai/animations/index.html
Gene overexpression methods (knock-ins, transgenics, reporter genes etc)
MODEL SYSTEMS
Proteomics
Nature (2003) March 13: Insight articles from pg 194
Analysis of protein expressionProtein structure and function Protein-protein interactions
Proteomics
Proteome projects - co-ordinated by the HUPO (Human Protein Organisation)
Involve protein biochemistry on a high-throughput scale
Problems limited and variable sample material, sample degradation, abundance, post-translational modifications, huge tissue, developmental and temporal
specificity as well as disease and drug influences.
Nature (2003) March 13: Insight articles from pgs 191-197.
Approaches in proteomics
Nature (2003) March 13: Insight articles from pgs 191-197.
High throughput approach
1)Mass- spectrometry
2) Array based
proteomics
3)Structural proteomics
High throughput approaches in proteomics
1) Mass spectrometry-based proteomics:Nobel prize in Chemistry (2002)
John B. Fenn
"for the development of methods for identification and structure analyses of biological macromolecules"
"for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules"
"for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological
macromolecules in solution
Koichi Tanaka Kurt Wüthrich
High throughput approaches in proteomics
1) Mass spectrometry-based proteomics: relies on the discovery of protein ionisation techniques.
used for protein identification and
quantification, profiling, protein interactions and modifications.
Nature (2003) March 13: Insight articles from pgs 191-197
two dimensional gels and mass spectrometry
Identification of proteins in complex mixtures
19_09.jpg
two dimensional gels
Mass spectrometry (MS)
Nature (2003) March 13: Insight articles from pgs 191-197
ionizer source: converts analyte to gaseous ions mass analyser: measures mass-to-charge ratio
(m/z)detector: registers the number of ions at each m/z
Principle of MS
Types of ionizer sources
Nature (2003) March 13: Insight articles from pgs 191-197.
Electrospray ionisation (ESI)matrix-assisted laser desortion/ionisation (MALDI)
MALDI-MS - simple peptide mixtures whereas ESI-MS - for complex samples.
2) Array-based proteomics
Nature (2003) March 13: Insight articles from pgs 191-197.
Based on the cloning and amplification of identified ORFs into homologous (ideally used for bacterial and yeast proteins) or sometimes heterologous systems (insect cells which result in post-translational
modifications similar to mammalian cells). A fusion tag (short peptide or protein
domain that is linked to each protein member e.g. GST) is incorporated into the plasmid construct.
Array based proteomics….
Nature (2003) March 13: Insight articles from pgs 191-197.
a. Protein expression and purification b. Protein activity: Analysis can be done using
biochemical genomics or functional protein microarrays. c. Protein interaction analysis two-hybrid analysis (yeast 2-hybrid), FRET (Fluorescence resonance energy transfer), phage display etc d. Protein localisation: immunolocalisation of epitope-tagged products. E.g the use of GFP or luciferase tags
Array based proteomics….
Nature (2003) March 13: Insight articles from pgs 191-197.
Protein chips
Antibody chips – arrayed antibodiesAntigen chips – arrayed antigensFunctional arrays – arrayed proteinsProtein capture chips – arrayed capture agents that interact with proteins e.g. BIAcoreSolution arrays – nanoparticles
19_14.jpg
3) Structural proteomics
8HDF / MTHF?
FAD
Modelling of a novel photolyase based on sequence Winnie Wu
Identification of protein-protein
interactions
affinity capture/mass spectrometry
Fig. 10. 31
Identification of protein-protein interactionsPhage display
Fig. 10.32
Systems Biology Systems Biology the global study of multiple components of the global study of multiple components of
biological systems and their interactionsbiological systems and their interactions
– Sequencing genomes– High-throughput platform development– Development of powerful computational
tools– The use of model organisms– Comparative genomics
New approaches to studying biological systems
19_20.jpg
Six steps in systems approach
• Formulate computer based model for the system
• Discovery science to define as many of the system’s elements as possible
• Perturb the system genetically or environmentally
• Integrating levels of information from perturbations
• Formulate hypothesis to explain disparities between model and experimental data
• Refine the model after integrating data
Nitin S. Baliga et al. Genome Res. 2004; 14: 1025-1035
Systems biology approach to studying how Halobacterium NRC-1 transcriptome responds to uv radiation
Challenges for the future – ‘
Genomics – CNV’s in medicine
‘Physiome’
Translational medicine
• General Reading– Chapter 19- HMG3 by Strachan and Read
Reference • Science 9 Feb 2007 Vol. 315. no. 5813, pp. 848 – 853
(CNV report)
• Nature (13 March 2003). Proteomics insight articles from Vol. 422, No. 6928 pgs 191-197
• Crit Rev Biotechnol. 2007 Apr-Jun;27(2):63-75 (good current review on challenges in transcriptomics /proteomics)
Resource:http://www.sanger.ac.uk/PostGenomics/