Transcript Gene expression and DNA microarrays • Old methods. • New methods based on genome sequence. – DNA Microarrays • Reading assignment - handout – Chapter 4

Gene expression and DNA
microarrays
• Old methods.
• New methods based on genome sequence.
– DNA Microarrays
• Reading assignment - handout
– Chapter 4 - 195-232, 239-250, 255 MIAME
– Box 4.1, 4.2 (Significance test only), 4.3, and
4.4.
Genome of the week
• E. coli O157:H7.
– Comparison to the laboratory strain of E. coli.
• Causes haemorrhagic colitis
– Initially identified in 1982 during an outbreak of severe
bloody diarrhea.
– Linked to contaminated ground beef from Michigan
– Can be lethal
• 75,000 cases per year
• Major findings:
– Comparison of E. coli O157:H7 with E. coli K-12
(common lab strain) found that the O157:H7 genome is
~ 1Mb larger than K-12 and contains 1,387 genes
specific for O157:H7.
– Genomes share a 4.1 Mb backbone with species
specific DNA interspersed throughout the genome
• K-islands - specific to K-12 (0.53Mb)
• O-islands - specific
– Lateral transfer of DNA occurs much more frequently
than previously thought. Especially high for
enterobacteria.
• O-island specific DNA encoded genes required for virulence
and a large number of phage and phage associated genes.
• Five sequences of E. coli - 4 pathogens and 1 lab
strain.
– Pathogens are as different from each other as they are
from the non-pathogenic lab strain.
Gene expression
• What is gene expression?
• Methods for measuring a single gene.
– Northern Blots
– Reporter genes
– Quantitative RT-PCR
• Operons, regulons, and stimulons.
• DNA microarrays.
– Expression profiling
– Identifying protein binding sites.
– Comparing gene content of different strains.
What is gene expression?
• The amount of RNA produced from a gene.
• Level of RNA produced from a gene is controlled
by:
– Transcription
– Stability/Degradation
• Transcriptome - Expressed transcripts in a cell
under defined experimental conditions.
– mRNA(5-10% of total RNA).
– rRNA, tRNA - make up most of total RNA
– scRNA (protein secretion), tmRNA (rescue stalled
ribosomes).
Regulons and Stimulons
• Operon - group of genes co-expressed on a single
transcript.
– One location of the genome
• Regulon - genes that are regulated by a single
transcription factor.
– Genes and operons throughout the genome
• Stimulon - collection of genes that are regulated in
response to environmental changes.
– Can be multiple regulons affected at once.
• Regulatory network - alternative term for regulon.
Analysis of gene expression at
the single gene level.
• Northern Blots
– Measure RNA levels by hybridization of a
labeled probe to total RNA.
• Reporter Genes
– Use of an enzyme to measure the amount of
transcription from a promoter.
• Quantitative real-time RT-PCR.
• Brief review in book.
Quantitative real time RT-PCR
Depth of knowledge
Challenges Facing Genomics
Detailed
analysis of
single gene
New tools in
genomics:
microarrays and
proteomics
Genome
sequencing
Breadth of knowledge
Assaying the regulation of 1000s
of genes in a single experiment
• DNA microarrays
– DNA molecules printed at high density used to
determine the level of RNA or DNA in a
sample.
– Can be thought of a “reverse Northern blots”
• Other technologies (described in chapter 4).
– SAGE
– Microbeads
DNA Microarrays -Introduction
• Spotted DNA arrays (glass slides)
– Competitive binding of samples - internal
control
– Fluorescent detection - Cy3 and Cy5
– Small sample sizes (10-30µl).
– PCR or cDNA arrays - double stranded
– Long oligonucleotide arrays - single stranded
• Better specificity, cheaper, easier to work with.
• Short oligonucleotide arrays
– ex. Affymetrix
• DNA spotted onto nylon membranes
(macroarrays)
Applications of DNA microarrays
• Expression profiling
– Determining the relative levels of RNA in two or more
samples.
• DNA/DNA hybridizations
– Investigate gene content between different strains
– Determine gene dosage
– 16S arrays - microbial communities (being developed).
• Identification of protein binding sites
– ChIP-Chip. Immunoprecipitation of protein/DNA
complexes. Assaying those interactions with
microarrays.
Uses of DNA microarrays
• Detection of candidate genes
– Expression profiling
– DNA/DNA hybridizations
• Annotation of gene function
– Expression - compendium approach
• Defining regulatory networks
– Expression profiling
– ChIP/chip experiments
• Molecular phenotyping
– Expression profiling
– DNA/DNA hybridizations
Microarray experimental overview
37C
25C
Grow cells
Isolate RNA
Make labeled cDNA
Mix and hybridize
Scan slide
Analyze data
Hybridization: basic concept
The ability of two strands to hybridize is
dependent on their complementarity.
More complementarity=better hybridization
Bacterial DNA microarrays
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Small genome size
Fully sequenced genomes, well annotated
Ease of producing biological replicates
Genetics
B. subtilis DNA microarrays
• PCR generated microarrays using custom primers
(Sigma-Genosys).
• Each PCR product represents a single gene.
• 4074 genes of 4101 on the array.
• Printed on Corning CMT-GAPS slides.
• 4 E. coli controls, each represented 15-20 times on
the array.
How a DNA microarray works
• Comparing the genome content of two B.
subtilis strains.
• The two strains differ only by the fact that
JH642 is lysogenized with the
bacteriophage SPb.
• JH642 vs PY79 genomic DNA
hybridization.
– PY79 does not contain SPb.
– SPb spots will be red.
JH642
PY79
Array size = 16mm x16mm
Spot size = 150mM
JH642
PY79
SPb genes
E. coli control