This document discusses DNA microarrays, including: 1. DNA microarrays contain many DNA probes attached to a solid surface that allow measurement of gene expression levels or genotyping of many regions simultaneously through hybridization. 2. The core principle is hybridization - complementary nucleic acid sequences pair through hydrogen bonds, and fluorescent labeling allows detection of binding to quantify expression. 3. DNA microarrays have many applications including gene expression profiling, disease diagnosis, drug discovery, and toxicology research.

1. DNA MICROARRAY
TECHNIQUES
PRESENTED BY ,
GAYATHRY P
M PHARM
DEPT OF PHARMACOLOGY
ACHARYAAND BM REDDY COLLEGE OF PHARMACY

2. INTRODUCTION
 A DNA microarray , also commonly known as DNA chip or biochip is a
collection of microscopic DNA spots attached to a solid surface.
It is to measure the expression levels of large numbers of genes simultaneously or
to genotype multiple regions of a genome.
 Each DNA spot contains picomoles of a specific DNA sequence known
as probes.
 Each known gene or probe occupies a particular spot on the chip and varying
levels of gene activity in introduced genetic material.
 Microarray use hybridisation to detect a specific DNA or RNA in sample.

3.  Fluorescently labelled target sequences that bind to probe sequence generate a
signal.

4. PRINCIPLE
 The core principle is HYBRIDISATION.
 The property of complementary nucleic acid sequences specifically pair with each
other by forming hydrogen bonds between complementary nucleotide base pairs.
 A high number of complementary base pairs in a nucleotide sequence means
tighter non-covalent bonding between the two strands.
 After washing off non-specific bonding sequences, only strongly paired strands will
remain hybridized.
 Fluorescently labeled target sequences that bind to a probe sequence generate a signal
that depends on the hybridization conditions (such as temperature).

5.  Total strength of the signal, from a spot, depends upon the amount of target
sample binding to the probes present on that spot.

6. REQUIREMENTS
1. DNA chip.
2. Target sample ( fluorescently labelled ).
3. Enzymes .
4. Fluorescent dyes.
5. Probes.
6. Scanner.

7. STEPS IN IMPLEMENTATION OF DNA MICROARRAY
SAMPLE
PREPARATION &
LABELLING
WASHING
IMAGE
ACQUISITION
& DATA ANALYSIS
HYBRIDISATION

8. SAMPLE PREPARATION AND SAMPLING
Isolate total RNA containing mRNA that ideally represents a
quantitative copy of genes expressed at the time of sample collection.
Preparation of cDNA from mRNA using a reverse transcriptase enzyme.
Short primer is required to initiate cDNA synthesis
Each cDNA ( sample and control )is labelled with fluorescent cyanine dyes

9. HYBRIDISATION
The labelled cDNA are mixed together
Purification
After purification the mixed labelled cDNA is competitively hybridised against
denatured PCR product or cDNA molecule spotted on glass slide

10. IMAGE ACQUISITION & DATAANALYSIS
Slide is dried and scanned to determine how much labelled cDNA ( probe ) is
bound to each target.
Hybridised target produce emissions
 Microarray software uses green spots on microarray to represent upregulated genes.
 Red represent those genes that are down regulated.
 Yellow represent equal abundance.

11. EQUIPEMENTS COLLECT TISSUE

12. ISOLATE RNA ISOLATE mRNA MAKE LABELLED DNA COPY
APPLY DNASCAN MICROARRAYANALYSE DATA

13. TYPE OF DNA CHIPS
1) cDNA BASED MICROARRAY
2) OLIGONUCLEOTIDE BASED MICROARRAY

14. cDNA BASED MICROARRAY
The probes are oligonucleotides, cDNA or small fragments of PCR products that
correspond to mRNAs.
 The probes are synthesized prior to deposition on the array surface and are then
"spotted" onto glass.
 A common approach utilizes an array of fine pins or needles controlled by a robotic
arm that is dipped into wells containing DNA probes and then depositing each probe
at designated locations on the array surface.
 The resulting grid of probes represents the nucleic acid profile.
 Solid made up of nylon filter or glass slide ( 1*3 inches).
 The cDNA is amplifies by using PCR.

15. OLIGONUCLEOTIDE BASED ARRAY
 probes are short sequences designed.
 Oligonucleotide arrays are produced by printing short oligonucleotide sequences
designed to represent a single gene or family of gene splice-variants by synthesizing this
sequence directly onto the array surface instead of depositing intact sequences.
 Sequences may be longer (60-mer probes such as the Agilent design) or shorter (25-mer
probes produced by Affymetrix ) depending on the desired purpose.
longer probes are more specific to individual target genes ; shorter probes may be
spotted in higher density across the array and are cheaper to manufacture.
One technique used to produce oligonucleotide array is ,photolithographic synthesis
(Affymetrix).

16. IN-SITU LIGHT DIRECTED OLIGONUCLEOTIDE PROBE
ARRAY SYNTHESIS
Light is directed through a photolithographic mask to specific areas of array
surface.
Activation of areas of chemical coupling . Attachment of A nucleotide
containing photolabile protecting group X
Next light Is directed to a different region of array surface through a new mask
Addition of second building block T containing a photolabile protecting group
X. This process repeated until the desired product is obtained.

17. PHOTOLITHOGRAPHIC SYNTHESIS
 It is on a silica substrate where light and light-sensitive masking agents are used to
"build" a sequence one nucleotide at a time across the entire array.
 Each applicable probe is selectively "unmasked" prior to bathing the array in a solution
of a single nucleotide, then a masking reaction takes place and the next set of probes are
unmasked in preparation for a different nucleotide exposure.
 After many repetitions, the sequences of every probe become fully constructed.
 Recently, Mask less Array Synthesis from Nimble Gen Systems has combined flexibility
with large numbers of probes.

18. cDNA Based Microarray Oligonucleotide Based Microarray

19. APPLICATION

20. 1. Gene expression profiling.
2. Disease diagnosis.
3. Drug discovery
4. Toxicological Research
5. Gene ID
6. Nutrigenomic research.
7. SNP detection.
8. Tiling Array.
9. Dam ID
10. Alternative splicing detection.

21. GENE EXPRESSION PROFILING
 Used to detect DNA ,RNA that may or may not be translated into proteins.
 The process of measuring gene expression via cDNA called expression
analysis
 Thousands of gene are simultaneously assessed .
 Study the effect of certain treatments , disease, and developmental stages on
gene expression.

22. DISEASE DIAGNOSIS
 Help to investigate about different diseases
• Earlier cancer classified on the basis of organs in which tumours develop.
• Now, classify the type of cancer on the basis of patterns of gene activity in
tumour cell.
• Help to produce very effective drug.

23. DRUG DISCOVERY
 Extensive application in PHARMACOGENOMICS.
 Comparitive analysis of genes.
 Help to identify specific proteins produced by diseased cell.
 Information used to synthesize drug which combact with these proteins and
reduce their effects.

24. TOXICOLOGICAL RESEARCH
 A rapid platform for the research of impact of toxins on the cells and
their passing on to the progeny.
 Important for Toxicogenomic studies.

25. GENE ID
Small microarrays to check IDs of organisms in food and feed mycoplasmas in
cell culture, or pathogens for disease detection.
 Mostly combining PCR and microarray technology.

26. NUTRIGENOMIC RESEARCH
 Study variation in genes related to the influence on diet.
 These variation , known as single nucleotide polymorphism.

27. THANK YOU