Gene Expression Analysis Unit 19

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Gene Expression AnalysisUnit 19

• BIOL221T Advanced Bioinformatics for
  Biotechnology

Irene Gabashvili, PhD
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Major challenge in biology

• Gain an understanding of the workings of the cell
  by integrating available information from the
  various fields of molecular and cellular biology
  and physiology into an accurate model to generate
  hypotheses for testing
• In previous lectures we mostly talked about
  Genome (Sequence) informatics

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-Omes -Omics

• Genome - all the genes of an organism
• Transcriptome all the transcripts (mRNAs) of an
  organism
• Proteome all the proteins of an organism
• Metabolome all metabolites (low molecular
  weight molecules participating in general
  metabolic reactions required for the maintenance,
  growth) of an organism

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Sequencing Successes
T7 bacteriophage completed in 1983 39,937 bp, 59
coded proteins Escherichia coli completed in
1996 4,639,221 bp, 4,293 ORFs Sacchoromyces
cerevisae completed in 1996 12,069,252 bp, 5,800
genes
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Completed sequences
1995 First complete bacterial genomes 2002
About 35 bacterial genomes
0.5-5 Mb hundreds to 2000 genes 1996 April
Yeast (Saccharomyces cerevisiae)
12 Mb, 5,500 genes 1998 Dec. -Worm
(Caenorhabditis elegans)
97 Mb, 19,000 genes 2000 March - Fly (Drosophila
melanogaster) 137 Mb,
13,500 genes 2000 Dec. - Mustard (Arabidopsis
thaliana) 125 Mb, 25,498
genes 2000 June Human (Homo sapiens) 1st rough
draft 2001 Feb 15/16 Human, working draft
3000 Mb, 35,00040,000 genes
Mouse, rat, chimp
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Bac- by Bac shotgun (public sequence)) Total
shotgun from the BAC ends (Celera)
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No prerequisites
Clone contig is a prerequisite
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Human Genome Organization
HUMAN GENOME
Nuclear genome 3000 Mb 25-35-40-65-80K genes
Mitochondrial genome 16.6 kb 37 genes
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Genes and gene- related sequences
Extragenic DNA
Two rRNA genes
22 tRNA genes
13 polypeptide- encoding genes
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Unique or moderately repetitive
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90
Unique or low copy number
Moderate to highly repetitive
Coding DNA
Noncoding DNA
Pseudogenes
Gene fragments
Introns, untranslated sequences, etc.
Tandemly repeated or clustered repeats
Interspersed repeats
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Human RNA genes (non-coding RNA transcripts)

• 100000 RNA genes in human genome (rough)
• rRNA
• tRNA
• Small nuclear RNA
• Small nucleolar RNA
• SRP RNA
• MicroRNA
• Antisense RNA
• Non-coding gene mRNA isoforms
• RNAs form transcribed pseudogenes

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Human pseudogenes
Non-processed pseudogenes
Processed pseudogenes
Contain introns Arise by duplications
Frequency of transfer depend on chromosomal
context (pericentromeral fragment are transferred
more often)
Do not contain introns Arise by
retrotransposition Frequency of transfer
depends on initial level of gene
expression (Highly expressed genes are
transferred more often)
Partial
Complete
Both types of pseudogenes are raw material for
evolution
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Molecular Biology Tools

• Northern/Southern Blotting
• Differential Display
• RNAi (small RNA interference)
• Serial Analysis of Gene Expression (SAGE)
• DNA Microarrays or Gene Chips
• Yeast two-hybrid analysis
• Immuno-precipitation/pull-down
• GFP Tagging Microscopy

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SAGE

• Every mRNA molecule is converted into a short
  (10-14 base), unique tag. Equivalent to reducing
  all the people in a city into a telephone book
  with surnames
• After creating the tags, these are assembled or
  concatenated into a long list
• The list can be read using a DNA sequencer and
  the list compared to a database to ID genes or
  proteins and their frequency

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SAGE
Convert mRNA to dsDNA Digest with
NlaIII Split into 2 aliquots Attach Linkers
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SAGE
Linkers have PCR Tagging Endonuclease Cut
with TE BsmF1 Mix both aliquots Blunt-end
ligate to make Ditag Concatenate Sequence
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Hybridization

• Nucleic acid hybridization is a fundamental tool
  in molecular genetics. It takes advantage of
  the complementary nature of double stranded DNA
  or RNA to the DNA or even RNA to RNA.
• Nucleic acid probes are used extensively in many
  different diagnostic tests.
• Hybridization is also used in cloning and PCR

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Principles of hybridization

• The addition of a probe to a complex mixture of
  target DNA. The mix is incubated under
  conditions that promote the formation of hydrogen
  bonds between complementary strands.
• Factors that affect hybridization characteristics
• Strand Length
• Base Composition
• Chemical environment

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Principles of nucleic acid hybridization
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Types of probes
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Stringency

• Strand length
• The longer the probe the more stable the duplex
• Base Composition
• The GC base pairs are more stable than AT
• Chemical environment
• The concentration of Na ions stablize
• Chemical denaturants (formamide or urea)
  destablize hydrogen bonds.

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Reassociation Kinetics

• When double stranded DNA is denatured by heat the
  speed at which the strands form double stranded
  DNA is due to the starting concentration of DNA.
  If there is a high concentration of complementary
  DNA then the time required will be reduced.
  Reassociation Kinetics is the speed at which
  complementary single strands form duplexes. Two
  parameters is Concentration (Co) and time (t) in
  sec. (Cot) This dictates that single copy genes
  hybridize more slowly than multicopy sequences.
  Therefore give weaker signals on a southern.

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Bioinformatics Hybridization Techniques

• Software tools to design probes, calculate
  melting temperature, GC content, stability,
  folding
• Tools to design Primers short DNA sequences used
  to initiate the synthesis of DNA
• Tools to design Probes sequences of DNA or RNA
  used to detect complementary sequences by
  hybridization

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Tools to design Primers, Probes cloning
strategies

• Matlab
• VectorNTI
• MacVector www.macvector.com/
• http//array.iis.sinica.edu.tw/ups/
• http//frodo.wi.mit.edu/
• http//genome.jouy.inra.fr/cgi-bin/CloneIt/CloneIt
  

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Tools to design Primers and Probes

• In-Silico PCR - search for a pair of primers
• http//genome.ucsc.edu/cgi-bin/hgPcr
• http//bioinfo.ut.ee/index.php?pid1
• http//bioinfo.ut.ee/mprimer3/
• http//bioinfo.ut.ee/genometester/
• http//bioinfo.ut.ee/maphdesigner/
• https//vectordesigner.invitrogen.com/

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Dot blot or slot blot
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Southern Blot
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Northern Blot
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Mutation detection by RFLP
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Assay of RFLP (restriction site polymorphism)
This has a variety applications including VNTR
RFLPs and DNA fingerprinting.
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Detection of gene deletions by restriction mapping
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In situ hybridization

• Chromosome in situ hybridization
• Metaphase or protometaphase chromosomes are
  probed with labeled DNA . The DNA can be labeled
  with a fluorochrome (FISH).
• Tissue in situ hybridization
• Sliced or whole mounted preparations can be
  probed with RNA probes to detect mRNA expression

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Hybridization Summary

• Hybridization is due to complementarity of DNA
  strands.
• DNA can be labeled various ways
• Hybridization can detect identical or similar
  sequences.
• A variety of techniques utilize hybridization of
  DNA or RNA probes Southern Blot, RFLP, VNTRs,
  Mutation detection, deletion detection, Northern
  Blot, tissue specific expression, In situ
  hybridization
• Microarrays are minaturized hybridization
  platforms

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DNA Microarrays

• Principle is to analyze gene (mRNA) or protein
  expression through large scale non-radioactive
  Northern (RNA) or Southern (DNA) hybridization
  analysis
• Brighter the spot, the more DNA
• Microarrays are like Velcro chips made of DNA
  fragments attached to a substrate
• Requires robotic arraying device and fluorescence
  microarray reader

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Microarrays

• Probe single-stranded DNA with a defined
  identity tethered to a solid medium Target
  the labeled DNA or RNA

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Microarrays
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History Types of Arrays

• The first arrays, created in the mid 80s, were
  called macro arrays. They were fabricated by
  spotting DNA probes on a membrane-type material
  with spot sizes of about 300 microns, which
  limited the density of the spots to about 2000
  probes. They mostly were used for DNA clones, PCR
  products or oligonucleotides and typically were
  used with radioactively-labeled targets.

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History Types of Arrays

• Next came microarrays, which were created by
  using pin spotters. These are pin-based robotic
  systems that can dispense an accurate volume of a
  DNA solution in a spot of about 150 microns onto
  a glass slide. DNA clones, PCR products or
  pre-synthesized oligonucleotides can be bound to
  the glass surface to create high-density arrays

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History Types of Arrays

• By the mid 90s, researchers were using 2 channel
  microarrays Templates for genes of interest were
  obtained and amplified by PCR. Following
  purification and quality control, aliquots were
  printed on coated glass microscope slides. Total
  RNA from both the test and reference sample was
  fluorescently labeled with either Cy3 or
  Cy5dUTP using a single round of reverse
  transcription. The fluorescent targets were
  pooled and allowed to hybridize, under stringent
  conditions, to the clones on the array.

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History Types of Arrays

• Rather than making arrays in the laboratory using
  spotters, oligonucleotides can be synthesized in
  situ on a surface, creating high-density arrays
  with up to 500,000 probe sequences. The first
  company to commercialize this type of technology
  was Affymetrix, which uses a proprietary
  light-directed oligonucleotide synthesis approach
  (Affy GeneChips).

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History Types of Arrays

• Agilent Technologies uses inkjet printing
  technology to build the oligonucleotides on
  standard format glass slides using
  phosphoramidite chemistry.
• Nanogen developed an electronic microarray,
  utilizing the natural charge of the DNA
• Illumina BeadChips - The Sentrix BeadChip
  technology is set up to perform multiple
  hybridizations in parallel. Probes 50mer
  oligonucleotides

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BO Chapter 16

• Annotating array probes
• Designing the Experiment
• Data Collection and Management
• Image processing
• Measures of Expression
• Normalization
• Finding Significant Genes

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BO Chapter 16, cont

• Expression Vectors
• Clustering Approaches
• Beyond Statistical Significance and Clustering
• The Classification Problem
• Distances
• Fisher Exact Test

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The starting point Annotating Array Probes

• Approaches to construct DNA arrays in-sity
  synthesis, randomly assembled bead-based arrays,
  mechanically spotted arrays (cDNA clone,
  PCR-amplified amplicon or other material)
• Annotation Resources SOURCE, DRAGON, DAVID,
  RESOURCERER, TIGR Gene Indices, EGO databases
  (some no longer exist, see ex. links)
• Mapping software tools IPA

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Designing the Experiment

• 2-color microarrays
• Plenty of RNA sample direct comparison with dye
  swap (flip dye pairs)
• Limited sample balanced block design
• More than 2 samples are compared Reference
  design (common reference needed)
• One color
• Power calculations for statistically significant
  measures of gene expression

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Bioinformatics of Gene Expression

• Data Collection and Management (MIAME, MAGE-ML)
• Estimating Background
• Measures of Expression (log ratio)
• Normalization (2-channel arrays)
• Filtering
• Finding Significant Genes
• Custering

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Internet Resources

• Expression Databases
• Array Express www.ebi.ac.uk/arrayexpress/
• CIBEX cibex.nig.ac.jp/
• Gene Expression Omnibus www.ncbi.nlm.nih.gov/geo/
  
• Annotation
• The Source database source.stanford.edu
• DAVID http//david.abcc.ncifcrf.gov/
• Gene Ontology Database, KEGG

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Internet Resources

• Expression Software
• http//david.abcc.ncifcrf.gov/
• BASE base.thep.lu.se
• Bioconductor bioconductor.org
• TM4 software http//www.tm4.org/
• SAM http//www-stat.stanford.edu/tibs/SAM/
• Cluster/Treeview http//bonsai.ims.u-tokyo.ac.jp/
  mdehoon/software/cluster/software.htm
• HCE http//www.cs.umd.edu/hcil/hce/
• http//ihome.cuhk.edu.hk/b400559/arraysoft_mining
  _specific.html

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Commercial Software

• Spotfire spotfire.tibco.com
• GeneSpring www.genespring.com/
• Partek Pro http//www.partek.com/
• IPA http//www.ingenuity.com/

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Beyond Statistics

• IPA looks for significant functional
  associations, GO- and literature based
  associations, canonical pathways, predefined and
  custom gene lists, creates networks, reconstructs
  significant processes, finds biomarkers

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IPA, How to

• Upload Data
• Analyze Gene Expression Data
• Compare Gene Expression Experiments
• o Interpret results
• Functions, Diseases, Pathways, Networks, Lists,
  Molecules
• o Explore Networks
• Highlight, Overlay, Merge, Export, Share

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Learning IPA

• Workshop in Stanford on April 21st
  http//lane.stanford.edu/howto/index.html?id_2608

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Learning IPA

• o Merging Networks
• Simple search for genes/proteins/chemicals
• Using Node View Pages
• QA
• Hands-on exercises

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From Previous Lecture

• Intermolecular Interactions
• Interaction and Pathway Databases
• Search and Explore in IPA (Simple search for
  genes/proteins/chemicals, Advanced Search for
  diseases, molecule types, locations)
• Finding interaction partners and closest path in
  networks, in IPA
• Quick functional assessments in IPA