Microarray and sds page

1. INTRODUCTION TO MICROARRAY………………………………………………………………….pg.3
2. TYPES OF MICROARRAY TECHNIQUES…………………………………………………………….pg.4
3. DNA MICROARRAY………………………………………………………………………………………….pg.5
4. STEPS INVOLVED IN PREPARATION…………………………………………………………………..pg.6
5. MICROARRAY ANALYSIS…………………………………………………………………………………..pg.7-9
6. APPLICATIONS OF MICROARRAY………………………………………………………………………pg.10-11
7. INTRODUCTION TO SDS-PAGE………………………………………………………………………….pg.12-13
8. PROCEDURE……………………………………………………………………………………………………..pg.14-17
9. APPLICATIONS…………………………………………………………………………………………………..pg.18
10. REFERENCES………………………………………………………………………………………………………pg.19 2

An array is an orderly arrangement of samples where
matching of known and unknown DNA samples is done
based on base pairing rules.
The sample spot sizes are typically less than 200 microns in
diameter usually contain thousands of spots fixed onto a
microscopic slide.
Tens of thousands of features can be fixed to a single
microarray chip, able to quantify thousands of gene
transcripts, indeed the entire human transcriptome in a
single experiment.
Specific pattern of gene expression under a given set of
experimental conditions can be studied. 3

 DNA microarrays, such as cDNA microarrays, oligonucleotide microarrays.
 Peptide microarrays, for detailed analysis or optimization of protein-protein
interactions.
 Tissue microarrays.
 Cellular microarrays (also called transfection microarrays).
 Antibody microarrays.
 Carbohydrate arrays (glycan arrays).
4

 DNA Microarrays are 2D solid surfaces to which ssDNA molecules/oligonucleotides have
been immobilized(called probes)
 Also known as DNA chip, biochip, gene chips, gene arrays, genome chips & genome
arrays.
 Thousands of ssDNA molecules are attached in fixed locations (spot).
 The microarray chip is incubated with a biological extract (cDNA or oligonucleotide) that
is labelled with fluorescent dye to undergo Hybridization.
 Thus a single microarray experiment produces thousands of data points, each of which is
a measure of the fluorescent label bound.
 These fluorescence intensities are indirect estimates from which scientists measure
the expression levels of large numbers of genes simultaneously.
 Current DNA microarray Methodologies:
• Immobilized single stranded cDNA molecules:
 long, non-synthetic and low density.(on glass slide or nylon membrane)
 Probe is spotted on slide. (appx. 6400 spots/site)
• Immobilized short oligonucleotide ssDNA:
 short, synthetic, high density substrate.(on silicon chips)
 Probe is synthesized on chip.(appx. 80,000 spots/site) 5

 Samples of mRNA collected are converted to cDNA.
 Fluorescent markers are attached to these cDNA
fragments.
 They are allowed to react with probes of the DNA
chip.(hybridization)
 Target DNA fragments with complementary
sequences bind to DNA probes.
 The remaining DNA fragments are washed away.
 The target DNA pieces can be identified by their
fluorescence emission by passing a laser beam.
 A computer is used to record the pattern of
fluorescence emission & DNA identification.
 This technique is very rapid & used for identification of
several DNA fragments simultaneously. 6

Method that makes use of gene chips. By using such chips to quantify
mRNA levels in different tissues or in individuals under different
treatments, tens or hundreds of specific genes which vary in relation to
the tissue or treatment can be identified.
mRNA molecules collected from both an experimental sample and a reference sample.
the reference sample  from a healthy individual.
experimental sample  individual with a disease like cancer.
The two mRNA samples are then converted into complementary DNA (cDNA).
Each sample is labeled with a fluorescent probe of a different color.
Experimental (cancer) DNARED
Reference (healthy) DNA GREEN
The two samples are then mixed together and allowed to hybridize with DNA probe.
7

The data gathered is used to create gene expression profiles, showing simultaneous changes in the
expression of many genes in response to a particular condition or treatment.
if there is equal expression in the two samples, spot yellow.
the expression in the experimental sample is lower than in the reference sample, spot  green.
expression of a particular gene is higher in the experimental sample than in the reference sample,
spot on the microarray red.
Following hybridization and washing, the microarray is scanned to measure the expression of each
gene printed on the slide.
8

 Analysis of gene expression:
• Examining expression during development or in different tissues.
• Comparing genes expressed in normal vs. diseased states
• Analyzing response of cells exposed to drugs or different physiological
conditions.
 Monitoring changes in genomic DNA:
• It helps in identification of mutations .
• In Examining genomic instability such as in certain cancers and tumors (gene
amplifications, deletions).
10

 Drug discovery:
• Comparative analysis of the genes from a diseased and a normal cell will help the
identification of the biochemical constitution of the proteins synthesized by the
diseased genes.
• The researchers can use this information to synthesize drugs which combat with
these proteins and reduce their effect. Advancement in pharmacogenomics.
 Disease diagnosis:
• chips have been designed to detect mutations in p53, HIV, and the breast cancer
gene BRCA-1.
 Toxicological Research:
• provides a robust platform for the research of the impact of toxins on the cells and
their passing on to the progeny.
• Toxicogenomics establishes correlation between responses to toxicants and the
changes in the genetic profiles of the cells exposed to such toxicants.
11

Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis.
Qualitative Analysis of Protein mixture.
Molecular weight and size of protein.
Composition of protein.
Purity of protein
PAGE: major component is water.
Provides solid support and matrix to hold protein during the electrophoresis.
Adjusting the amount of acrylamide used in gel, migration of proteins can be
controlled.
More acrylamide, more hindrance for protein migration.
12

SDS PAGE- separation /migration takes place based on molecular mass.
SDS detergent has negatively charged dodecyl sulfate ion.
Prior to electrophoresis the disulfide bonds of protein is reduced making the structure less compact and
linear.
The ions form complex with proteins, resulting in an unfolding of proteins.(remove H bonds)
Amount of detergent complexed is proportional to mass of protein.
Larger protein mass  more detergent forms complex.
Proteins in a solution of SDS is overwhelmed by the dodecyl sulphate ions complexed with it and attain a net
negative charge proportional to their mass.
Mass to charge ratio of all proteins is same.
Hence, Separation of different molecular mass is based on concentration of acrylamide.
More the acrylamide conc. smaller the pore size
More difficult for larger proteins to migrate relative to small proteins  Molecular sieving effect.
13

REQUIREMENTS:
 PROTEIN SAMPLE (prokaryotes/ eukaryotes/ virus/ environmental sample etc)
 BUFFER SOLUTION : SDS, β-mercaptoethanol, Bromophenol blue, Glycerol.
 SEPARATING GEL soln. : pH 8.8
 STACKING GEL soln. : pH 6.8 both contain acrylamide(polymer chain) and bis-acrylamide(cross linking).
 GLASS PLATE.
 ELECTRODES.
 RUNNING BUFFER SOLUTION. (Tris-Glycine Chloride.)
14

SAMPLE PREPARATION:
 Add the buffer to protein sample.
 Heat at 95 degree celcius for 5 mins.
 SDS denatures the protein by disturbing H- bonds, hydrophobic and ionic
interactions.
 β-mercaptoethanol cleave disulfide bonds causing denaturation of
protein.
 Also SDS attaches to protein at every 2 amino acid residue together this
treatment unfolds and makes protein linear with a net negative charge.
(enabling separation of proteins based on their mol. Weight)
GEL PREPARATION:
 Gel polymerized between two glass plates kept vertically.
 Separating gel poured first, allowed to solidify and followed by stacking
gel solution addition.
 Plastic comb placed to form sample wells.
 Comb removed upon solidification.
Note: stacking gel ensures arrival of all proteins to separating gel at same
time.
15

ASSEMBLING:
 Gel cassette placed vertically between 2 electrodes.
 Positive electrode at bottom and negative on top.
 Insert into chamber Tris-glycine chloride buffer soln. added to allow conduction of current.
 SDS is present in gel and in running buffer to ensure denaturation & negative charge
throughout process.
SAMPLE APPLICATION:
 Sample pipetted into well.
 Bromophenol blue help visualize sample in well and glycerol increases sample density and
ensure sample falls to bottom of well.
MIGRATION:
 Electric field applied across gel.
 Negative charged protein move towards bottom positive electrode away from negative
electrode.
 First into stacking gel slowly and reach separating gel where they move freely.
 But separate as per their molecular wt., lower wt. move faster.
 Analysed by protein staining (coomassie blue+acetic acid) method and immunoblotting
detection.(western blotting)
16

1.Measuring molecular weight.
2.Peptide mapping.
3.Estimation of protein size.
4. Estimation of protein purity.
5. Monitoring protein integrity.
6. Protein quantification.
7. Comparison of the polypeptide composition of different
samples.
8. Analysis of the number and size of polypeptide subunits.
9. Post-electrophoresis applications, such as Western
blotting.
18

1. IMTIYAZ ALAM KHAN. ELEMENTARY BIOINFORMATICS. PharmaMed Press; p.p. 243-245
2. DAAN J. A. CROMMELIN, ROBERT D. SINDELAR, BERND MEIBOHM. PHARMACEUTICAL BIOTECHNOLOGY:
Fundamentals and Applications. 3rd ed. INFORMA HEALTHCARE; p.p.41-42, 138-139
3. DARRYL LEON, SCOTT MARKEL. IN SILICO TECHNOLOGIES IN DRUG TARGET IDENTIFICATION AND VALIDATION.
6TH ed. TAYLOR AND FRANCIS GROUP; p.p.123-124
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