02 microarray

7/27/2019 02 MICROARRAY

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MICROARRAYS

Created b : rou 2


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Agatha C. MaturbongsSylvia Hanna Sirait

Eko Prasetya

Yusnaeni Yusuf

Umi Kulsum Nur Q.

Atika Okta Melisa

Nurhening

Rinaldi Rizal


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Microarray or DNA chip, Is a assembly of oligonucleotide or other DNA

probes fixed on a small, fine grid of surface A microarray used to analyze the expression

of many genes at the same time One approach is to use cDNA preparad from

mRNA or to recognize sequence variation ingenes


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Each dot in the microarray pictured here consists of anoligonucleotide fragment of a single gene bound to a glass slide. Geneexpression of two types of cells (e.g., one wild-type and one mutant)

can be compared by labeling the cDNA from each cell


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Schematic drawing of a DNA chip


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Computer Analysis


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What are the underlying microarraytechnology?

Gene expression

Gene expression is a highly complex and tightly

regulated process that allows a cell to respond

dynamically both to environmental stimuli and to itsown changing needs.


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1. Transcription2. Messenger RNA3. Polyadenylation

4. Reverse Transcription5. cDNA6. Oligonucleotide

Synthesis

7. Nucleotide acidHybridization

8. Fluorescence9. DNA labelling

Is the process, by which the contained in a section of

DNA, is transferred to a newly assembled piece of mRNA


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Synthesis



Is transcrip from DNA template, and carries coding

information to the site of protein syntethis


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Synthesis



Is the covalent linkage of a polyadenylyl moiety, to a

mRNA molecule at 3 end


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Synthesis



Is the transferred of information from RNA to DNA, or the

reverse of normal transcription


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Synthesis



Is DNA syntesiszed, from a mature RNA template in a

reaction catalyzed by enzym reverse transcriptase


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Synthesis



Is the non biological chemical synthesis of defined short

sequence of nucleic acids


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Synthesis



Hybridization is a process of combining complementary

single strand nucleic acids into a single molecule


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Synthesis


8. Fluorescence9. DNA labelling It is a functional group in a molecule which with absorb

energy of a spesific wavelength and re-emit energy at adifferent wavelength


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Synthesis



Is an addition of chemical into DNA strand to make them

visualizable


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Scientists are using microarray technology totry understand fundamental aspects of

growth and development. Using microarray:

1) to analyse the mRNAs in cell, to reveal theexpression patterns of protein2) To detect genomic DNA sequence, toreveal absent or mutated genes


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1. Nucleic acid microarray, : gene expression,SNP detection, and genomic hybridization

2. Protein microarray : marker detection andpeptide array3. Tissue array: molecular biology and

immunohistochemistry4. Other molecule array : chemical array


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1. Investigating cellular states and processes2. Comparison of related species3. Diagnosis of genetic disease4. Genetic warming signs5. Precise diagnosis of disease6. Drug selection7.

Determination of gene function8. Pathogen resistance9. Following temporal variations in protein

expression


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ANALYSIS OF MICROARRAY DATA

Initial goal

To known Gene

expression


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ANALYSIS OF MICROARRAY DATA

Green spot only one targethybridize

Red spot only other target

hybridize Yellow spotboth hibrydize

Raw data from microarray experiment

Is shown by different fluorescent spot

Data from many spot on themicroarray will contribute to

the calculation of the relative

expression level of each gene


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Principal uses of chips

Genome-scale expression analysis

Differentiation

Response to environmental factors

Disease states

Effect of drugs

Detection of sequence variation


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Comparison focused on the

genes

Comparison focused on the

samples

General approach to analize

matriks gene

expression

M i ili i f diff


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+ - +

+ - - +

+ - - -

+ + -

- + -

Database(s)

1 2 3 4

Timepoints

Condition

1 2 3 4

Gene

A

B

C

E

D

0 60 120 180

Time

3 A C G G G C ATG 5

3 A C G G G C ATG 5

3 A C G G G A ATG 5

Local Alignment

Search Window

A

C B-

0

+

Measure similiarity of different

rows & columns


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Result of similiarity analize of gene

expression matrix ofthen display ed

as a chart, coloured according to the

difference in expression pattern


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Microarry Data Variablity

Microarray data are inherently highly variable- you are measuring mRNA levels

Any kind of measurement of thousands ofvalues across 2 samples will find some largedifferences due to chance (normaldistribution)

Must have replication and statistics to showthat differences are real


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Higher Level

Microarray data analysis

Clustering and pattern detection

Data mining and visualization

Controls and normalization of results Statistical validatation

Linkage between gene expression data and gene

sequence/function/metabolic pathways databases

Discovery of common sequences in co-regulated

genes

Meta-studies using data from multiple experiments


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Expression Patterns in

Different Physiological States


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How different components of cells smoothly integrate their

activity?

Alternative physiological states of an organism offer the

possibility of extracting (from an entire genome) a subset of

genes that underlie a particular life process.

Effect of the reconfiguration of the expressions patterns,

different of genes active in the two states.

In yeast : Saccharomyces cerevisiae

Diauxy.Fermentative Oxidative metabolism

In rats and fruit flies (higher organisms)

Changes in gene expressions patterns in sleep and wakefulness.


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The diauxic shift in Saccharomyces cerevisiae

Capability of adapting metabolism to a variety of

environmental conditions.

Presence of glucose :

Metabolic anaerobic

Glucose Ethanol,

via glycolysis and fermentation(Emden-Meyerhof pathway).

Exhaustion of glucose, leads diauticshift :

Metabolic aerobic

Ethanol CO2 and H2O, via theKrebs cycle and oxidativephosphorylation.


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Figure 5.3 Some metabolic pathway in yeast affected by the

diauxic shift


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The shift (different source of energy)

creates two concomitant problems :

The oxidation of ethanol doesnt

provide precursor for essential

biosynthetic pathways.

Yeast must activate pathways of

defense against oxidative stress.


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High glucose :

Mig1 (in cytoplasm) , upon dephosphorylation enters the nucleus

and binds to promotors of various genes repressor.

(lot of glucose increase ATP kinase inactive Mig1

active repression of glucose-sensitive genes)

Glucose acts as a repressor, but when

its exhausted, this repression will escapeturning on a variety of genes.


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Image captured by scanner


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Expression ratio in aerobic/anaerobic states > 2 < > 4 < Number of gene 710 1030 183 203


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Upon converting from anaerobic to aerobic metabolism,

several things occur :

a. Metabolic pathways are rerouted

b. Genes related to protein show a decrease in expression

level

c. Genes related to a number of biosynthetic pathways

show reduced expression.

d. Genes involved in defense against oxidative stress from

ROS show enhanced expression.


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The characteristics of sleep are:approximately cyclic periods of reducedconsciousness, relaxation, and quiescence.

Sleep correlate with changes in brainelectrical activity. Comparison of flies and mammals may reveal

fundamental common features disguisedwithin the individual complexities of differentspecies.

Cirelli and co worker studied gene expression


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Cirelli and co-worker studied gene expressionpatterns of rats and fruit flies in three

physiological state:

1. spontaneously awake2. spontaneously asleep

3. sleep deprived


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Cirelli and co-workers studied expressionpatterns of ~ 10000 genes of the fly.

Of these, 121 were wakefulness related and

12 were sleep related. The expression of a partly overlapping set of

130 genes was moderated by time of day: 87

were more highly expressed at 4 p.m. and 43were more highly expressed at 4 a.m.

I b th fli d t th f ti ll


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In both flies and rats, the genes preferentiallyexpressed in wakefulness and sleep fell into

several different functional categories:

In flies, gene preferentially expressed in wakingstates included those encoding proteins involvedin detoxification, gene involved in defences

against immune challenge and in lipid,carbohydrate, and protein metabolism.

Genes preferentially expressed in sleep included

the glial gene anachronism, the gene encodingthe catalytic subunit of glutamate-cysteineligase and other genes involved in lipidmetabolism.

In rat brains, a much larger number of


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In rat brains, a much larger number ofgenes than in flies had raised expressionlevels during wakefulness.


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Microarrays made possible a more systematicand thorough study for transcription patternduring stages its life.

Drossophilla melanogasterhas 4028 genes (1/3 oftotal).

86 % or 3483 are most of the genes testedchanged expression levels significantly at some

stages of life. There are 3219 genes exhibited at least a

fourfold difference between their highest andlowest levels of expression.


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The expression pattern of the eyes absentmutant, there are 33 genes and 11 genes are

already known the function in eyedifferentiation or phototransduction


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To identify pattern of gene reexpression duringdevelopment, applied a peak-binding algorithmto each genes expression profile. The result are:

36.3% showed a single major peak of expression. 40.3% showed two peaks in expression :

Early in embriogenesis and elevated pupation

Late in embriogenesis and continuing into the adultstage

23.4% showed multiple peaks in expression


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Both embrionic and pupal stage , forming and

physical activity is minimal Larval and adult stage have a more active

lifestyle but statis of anatomical form


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Blue indicated low level Yellow indicated high level

E : embryos L : Larvae P : Pupae A : Adults


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Learning and memory involve changes in thestructur and biochemistry ofnerve cell


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Fact about learning and memory:The observation that memory survives periods

of coma, during which neural activity ceases,proves this.


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Is a neural phenomenon underlying learningand memory.

Is a persistent increase in strength of asynaptic connection as a result of stimulationof the upstream cell.


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Bliss and Lomo (1973):That high-frequency stimulation of a synapse

during a finite time interval produced apersistent subsequent enhancement of thepost-synaptic response


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Learning must be regarded as a specialyzedform a development


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Change expression patterns of gene can because response to LTP

Park & co-workers: Studied genes that

change their expression pattern in responseto LTP. They studied cells from the mousedentate gyrus, a structure within the

hippocampus. The hippocampus is known to be involved inmemory formation, especially spatialmemory.


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London: metropolitancity

Taxi drivers must have an exhaustiveknowledge of the metropolitan

geography

About of optimal route betweenpoints both famous dan obscureFACT about LondonTaxi Drivers

Brain scans show that London

taxi drivers have a large

hippocampus than a controlgroup; and

The hippocampus enlarges with

time spent behind the wheel


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Following high-frequency stimulation for fourseparated 1-second intervals, total RNA wasextracted after several time intervals 30, 60,

90, and 120 minutes after stimulation and thenreverse transcribed into cDNA and hybridizedonto Affymetrix GeneChip arrays.

The chip reported 12000 genes and ESTs. Sample of unstimulated tissue provided controls.


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Within all time points, 1664 genes withstatistically significant changed expressionpatterns.

Of these

The genes identified suggest that LTP producedchanges in a variety of processes affecting cellmorphology and affects interactions amongcells and between cells and the extracellularmatrix

39% upregulated

61% downregulated

Functional Category Upregulated Downregulate


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d

The extracellular matrix and its regulation + +

Membrane protein/cell structure/adhesion mol + +

Neurosteroid hormone metabolisme - +

Cytokine/growth factor/reseptor + +

Other receptors/signalling + +

Ion channel + +

Transcription factor/regulation + +

Translation + +

Neurotransmitter receptor/neuromodulator + +

Regulation of cytoskeleton + +

Mitochondrial/energy production + +

Proteases/proteases inhibitors + +

Immunoresponsive proteins/oxidative stress... + +

Myelin-related proteins - +

Chromatine structure + -


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Transglutaminase is known to be expressed inneural tissue and appears in synapses. However,it was not known to be implicated in LTP.

Cystamine is an inhibitor of tranglutaminase andalso causes disulphide exchange productingunfolding.

Transglutaminase also helps to produce proteinaggregates in Huntingtons disease. Cystamine does ameliorate Huntingtons disease

in the mouse, but the mechanism is unclear


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Is one puzzling gene Use of a specific inhibitor of CDC25B protein

product blocked LTP.


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Genes with common time profiles Events happening at particular times


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Comparison of the distributions of homologuesin the genomes of rats, humans, Drosophila, andCandida elegans suggest that the culstering is

concerved in a evolution. The clustering may contribute to a mechanism

for common regulation of expression,

reminiscent of a bacterial operon.


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There are a significant difference between human and

chimpanzee

The set of genes that show different expression between

human and chimpanzee is rich in transcription factors. The differences in expression pattern are not uniform in

different tissues. Changes in expression pattern tend to be lower in X and

highly in Y chromosomes than in autosomes

Duplicated genes tend to show a higher divergence inexpression pattern non-duplicated genes.

The differences in expression pattern are not uniform, even

across different autosomes


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Specific Tissue Nucleotides Expressed genes


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Area of brain Function in human Function in chimpanzee if

known to differ from human

Dorsolateral prefrontalcortex

Anterior cingulated cortex

Brocas areas

Central part of cerebellum

Caudate nucleus

Pre-motor cortex

Area homologues to brocas

right hemisphere

Important for higher brain functions:working memory, conscious control ofbehavior

Autonomic functions: heart rate and bloodpressure, and cognitive functions such asreward anticipation, decision making,

empathy, and emotion

Mainly language

Coordinating complex movements such aswalking

Regulation and organization of informationsent to frontal lobes

Sensory guidance of movement ,activating proximal and trunk muscles

Not entirely clear, ome involvement incommunications without syntactic

contribution to language use

Gesture, especially control over

orofacial action, including

communicative acts


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Development of antibiotic resistance in

bacteria Childhood leukaemias


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Study in vancomycin, one of most powerfulantibiotics available for use in humans

Vancomycin is a 1,5 kDa glycopeptideantibiotic isolated from a soil bacterium inBorneo,Amycolatopsis orientalis

First used clinically in 1958


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1941 first clinical use of penicillin G 1942 appearance of penicillin-resistant Staphylococcus aureus 1950s multidrug-resistant S.aureus widespread 1956 vancomycin described 1958 first clinical use of vancomycin

1960 first clinical use of methicilin 1961 appearance of methicillin-resistant S. Aureus 1960s spread of methicillin-resistant S. Aureus 1970s Methicillin-resistant S. Aureus widespread 1988 appearance of vancomycin-resistant enterococci 1992 laboratory transfer of high-level vancomycin resistance from enterococci

to methillin-resistant S.aureus 1977 appearance of vancomycin-intermediate S. Aureus in clinical setting 2002 appearance of vancomycin-resistant S. Aureus in clinical setting

Pfeltz, R.F. & Wilkinson, B.J. (2004). The escalating challenge of vancomycin resistance in Staphylococcusaureus. Curr. Drug Targets Infect. Disord.4. 273-294


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Resistance of S. Aureusstrain

Minimum inhibitoryconcentration (MIC)

Year first appeared

Sensitive 1 g/ml -

Intermediate (VISA) 8-16 g/ml 1997

Resistant (VRSA) >32 g/ml 2002


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Mongodin et.al (2003) : compared expressionpatterns of genes in VISA strains (MIC8g/ml)with VRSA strains produced by selection

The array contained 2688 oligonucleotides The experiment were run in parallel, starting with

two different clinical VISA isolates 35 genes consistently showed increased

expression, some as high as 30-fold

16 genes consistently decreased expression Many of the change of expression levels combine

to funnel metabolites to formation of ATP


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The novel development associated withgenomics is possible to identify targets-

specific proteins, essential for pathogen, that

differ from mammalian proteins sufficientlyto suggest that drugs against these proteinswould be effective against the pathogen but

non-toxic to mammals


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Leukaemia is the uncontrolled proliferation ofany blood cell at any stage of differentiationcausing by an abnormal genetic transformation

Leukaemias can be classified according to thetype of cell that proliferating :- acute lymphoblastic leukaemia- development of second cancer, is a common

and very serious complication of acutelymphoblastic leukaemia, for.i : braintumour


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In a study using 14.500 probe sets and samples from173 patient, sets of 20-40 genes were identified thatdistinguish resistance and sensitivity to four

different drugs : prednisolone, vincristine,

asparaginase, and daunorubicin at the genetic level reveals that the activities of the

drug involve some different pathways

A set of 45 genes was found to be correlated with

resistance to all four of the drugs The majority of these genes involve transcription,

DNA repair, cell-cycle maintenance and nucleic acid

metabolism


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Microarray technology enable to compare the activities ofthousand of genes in cell types from various tissuesamples in normal and diseased cell.

Expression pattern can predict effectiveness of treatmentand guide the choice of therapy.

Identification of specific genes involves in disease cansuggest targets for drug development, for instance ; onetype of acute lymphoblastic leukaemia is associated withoverexpression of the gene FLT3 for a receptor tyrosinekinase

By the knowledge of the type of leukaemia cancer, the

phycicians can choose the treatment that will probably bemost effective for that patient

Examining patterns of gene activity can identify whichpatient will probably remain free of cancer after treatmentand which will probably relapse.


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02 microarray

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