this presentation contains application and working of the microarray.

1. SUBMITTED BY
PRATEEK KUMAR

2. 1. Amount of mRNA expressed by a gene.
gene expression array, exon array, tiling array
2. Amount of mRNA expressed by an exon.
exon array, tiling array
3. Amount of RNA expressed by a region of DNA.
tiling array
4. Which strand of DNA is expressed.
exon array, tiling array
5. Which of several similar DNA sequences is present in the genome.
SNP array
6. How many copies of a gene is present in the genome.
gene expression array, exon array, tiling array
7. Where a known protein has bound to the DNA. (ChIP on chip)
promoter array, tiling array

3. ◦ Identify Genes expressed in different cell types (e.g. Liver vs
Kidney)
◦ Learn how expression levels change in different developmental
stages (embryo vs. adult)
◦ Learn how expression levels change in disease development
(cancerous vs non-cancerous)
◦ Learn how groups of genes inter-relate (gene-gene
interactions)
◦ Identify cellular processes that genes participate in (structure,
repair, metabolism, replication, … etc)

4.  • Gene discovery
◦ - tissue profiles
◦ - time course data
- altered genetic backgrounds
 • Comparing tissues/genotypes
 there’s a lot of promise in medicine (especially cancer research)
for this

5. • Microarray technology can be combined with other methods
for purposes in addition to looking at transcription
(“transcriptional profiling”). For instance, it can be used
along with chromatin immunoprecipitation (ChIP) to look at
proteins bound to DNA within the cell. This works best with
whole genome tiling arrays and be used to look at
transcription factor binding and post translation
modifications to histone proteins.

6.  Protein-binding microarrays can be used to identify
transcription factor binding sequences (motifs)
• double-stranded DNA probes used on array
• purified protein hybridized to array
• detected by antibody to protein or to epitope tag
• can use real genomic sequence or carefully designed
oligonucleotides
• possible to look at all possible 10-mer nucleotide sequences
on a single array

8. cDNA “A”
Cy5 labeled
cDNA “B”
Cy3 labeled
Hybridization Scanning
Laser 1 Laser 2
+
Analysis Image Capture

10. DNA microarrays, microscopic arrays of large sets of DNA sequences immobilized
on solid substrates, are valuable tools in areas of research that require the
identification or quantitation of many specific DNA sequences in complex nucleic
acid samples . These arrays can be assayed for changes in the expression patterns
of the representative genes after different treatments, different conditions or tissue
sources.
APPLICATION OF MICROARRAY IN MEDICINE

11. Microarrays, based on Southern's method of nucleotide hybridization,
contain multiple DNA sequences (probes) spotted or synthetized on a
relatively small surface. This feature of microarrays allows the
simultaneous monitoring of the expression of thousands of genes, thus
providing a functional aspect to sequence information, in a given sample
Currently, genomic microarrays are used in medicine for the following
purposes:

12. 1.Determination of transcriptional programs of cells for a
given cellular function (e.g., cell function, cell differentiation,
etc.) or when they are exposed to certain conditions leading to
activation, inhibition or apoptosis.
2.Compare and contrast transcriptional programs to aid
diagnosis of diseases, predict therapeutic response and
provide class discovery and sub-classification of diseases.
3.Identification of genome-wide binding sites for
transcriptional factors that regulate the transcription of
genes.
4.Prediction of gene function

13. 5.Identification of new therapeutic targets (target identification, target validation,
and drug toxicity).
6. Genetics of gene expression: Although this is a relatively new study field, it is
advancing rapidly with major implications in complex clinical traits by the
identification of promising candidate genes. Thus, we briefly review the current
implementations of this novel approach highlighting its necessity in the research
field. Treating mRNA transcript abundances as quantitative traits and mapping
gene expression quantitative trait loci for these traits has been pursued in gene-
specific ways.

14. STEP 1: Print or purchase a preprinted DNA microarray Scientists
doing a microarray: Prepare DNA microarrays by selecting and
“printing” known sequences of DNA (approximately 20-70 bases long)
that represent specific genes from an organism. Because “printing” a
chip is a very time-consuming process, most scientists order pre-
printed microarrays from companies that automate the microarray
printing process.
ANALYSIS OF CANCEROUS AND NON
CANCEROUS CELLS THROUGH
MICROARRAY:

15. Step 2: Collect cancerous colon cells and normal colon
cells from a patient.
Identify genes involved in colon cancer, by comparing
cancerous colon cells with normal (control) colon cells
from the same patient.
STEP 3: Isolate mRNA from two types of
cells
Isolate the messenger RNA from normal cells and from
cancer cells. when a gene is expressed in a cell, the DNA is
transcribed (copied) to make messenger RNA molecules.

16. STEP 4: Use reverse transcriptase to synthesize cDNA from
mRNA
Treat the messenger RNA from both cell types with reverse transcriptase,
an enzyme that copies the base sequence on mRNA molecules to make
complementary, single-stranded DNA known as cDNA (complementary
DNA) molecules.
STEP 5: Label the cDNA’s
Label the cDNA’s from the two kinds of cells with different colors of
fluorescent dye. For example, scientists may label the cDNA from cancer
cells with red dye and the cDNA from normal cells with green dye.

17. STEP 6: Mix the labeled cDNA’s
together
Mix together the labeled cDNAs from the normal and cancer cells in a single
test tube. This mixture is called a “hybridization solution.”
STEP 7: Hybridize the microarray with
labeled cDNA’s.
Soak the microarray in the hybridization solution (mixture of labeled cDNAs).
The cDNAs that are complementary to the DNA (gene) sequences on the
microarray spots will hybridize (bind) to the spots.
STEP 8: Remove unbound (unhybridized)
cDNA’s
Wash the microarray to remove the cDNA strips that are
not hybridized to gene spots.

18. 9.Scan to make the microarray
Scan the microarray with two laser lights that cause the fluorescent
labels on the cDNA to emit colored light. Scanning with one laser
causes the cDNA from cancer cells to emit red light. Scanning with a
second laser causes the cDNA from normal cells to emit green light
Step 10: Computer Merging of Images
Use a computer to merge the red and green scanned images. If both
red and green labeled cDNA are bound to the same spot, this results in
a YELLOW spot. If no cDNAs are bound to a spot, this will result in
a black spot.

19. Step 11: Analyze the results of the
gene expression study
The microarray color pattern is analyzed to identify which genes
are expressed differently in the two types of cells and which
genes might be involved in causing or preventing colon cancer

20.  Microarrays can measure the expression of
thousands of genes simultaneously
 Vast amounts of data require computers
 Types of analysis
◦ Gene-by-gene
 Method: Statistical techniques
◦ Categorizing groups of genes
 Method: Clustering algorithms
◦ Deducing patterns of gene regulation
 Method: Under development

21. Thank you