APPLICATIONS OF MICROARRAY

SUBMITTED BYPRATEEK KUMAR

1. Amount of mRNA expressed by a gene.

gene expression array, exon array, tiling array2. Amount of mRNA expressed by an exon.

exon array, tiling array3. Amount of RNA expressed by a region of DNA.

tiling array4. Which strand of DNA is expressed.

exon array, tiling array5. Which of several similar DNA sequences is present in the genome.

SNP array6. How many copies of a gene is present in the genome.

gene expression array, exon array, tiling array7. Where a known protein has bound to the DNA. (ChIP on chip)

promoter array, tiling array

What Can be Measured using Microarrays?

◦ 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)

• 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

• 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.

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

Expression profiling with cDNA microarrays

cDNA “A”Cy5 labeled

cDNA “B”Cy3 labeled

Hybridization Scanning

Laser 1 Laser 2

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Analysis Image Capture

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

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:

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

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.

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:

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.

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.

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.

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.

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

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

Analysis of microarray data

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