DNA MICROARRAY AND ITS APPLICATION

Ananta K. Ghosh Department of Biotechnology Indian Institute of Technology, Kharagpur

WHAT IS AN ARRAY ? An array is an orderly arrangement of samples. It provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns. An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample. Arrays can be of two types: macroarrays or microarrays.the difference being the size of the sample spots. Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots.

DNA MICROARRAY
It consists of large number of known cDNA or oligonucleotides spotted onto a carrier in a logical and orderly fashion.

DNA microarray, or DNA chips are fabricated by high-speed robotics, generally on glass but sometimes on nylon substrates, for which probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies.

An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously - a dramatic increase in throughput.

APPLICATION OF DNA MICROARRAY

Two major application forms for the DNA microarray technology:

Determination of expression level (abundance) of genes.

2) Identification of sequence (gene / gene mutation)

GENE EXPRESSION Gene expression is the term used to described the transcription of the information contained within the DNA-the repository of genetic information-into messenger RNA molecules that are then translated into the proteins that perform most of the critical functions of cells.

Gene expression is a highly complex and tightly regulated process that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs.

This mechanism acts as both an on/off switch to control which genes are expressed in a cell as well as a volume control that increases or decreases the level of expression of particular genes as necessary.

ANALYSIS OF GENE EXPRESSION

Thousands of genes and their products (i.e., RNA and proteins) in a given living organism function in a complicated and orchestrated way that creates the mystery of life.

However, traditional methods in molecular biology (northern hybridization or Biochemical approaches) generally work on a "one gene in one experiment" basis, which means that the throughput is very limited and the "whole picture" of gene function is hard to obtain DNA sequencing programme unravels the complete genome sequences of many organisms and identifies many new genes. But only after the full functions of the new genes are discovered the full impact of these genome project can be realized.

ANALYSIS OF GENE EXPRESSION DNA sequencing programme unravels the complete genome sequences of many organisms and identifies many new genes. But only after the full functions of the new genes are discovered the full impact of these genome project can be realized. And scientists are looking for different technologie. to address this issue

DNA Microarray is one such technology which allows the simultaneous analysis of thousands of genes and their functions

This technology promises to monitor the whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously.

TECHNOLOGY FOR MICROARRAY

Two major technologies are currently used for DNA microarray

A. cDNA microarray : It was developed by Brown and Schena at Stanford University and comprises a large number of cDNA (500 5000 bases long) spotted on to a glass slide, in a high density pattern and exposed to a set of targets either separately or in a mixture.

B Oligonucleotide array or chips: It was developed by the leading manufacturer, Affymatrix, and is made from oligonucleotides (20-80 nucleotides) array synthesized in situ on the glass surface by photolithiography or by conventional synthesis followed by on chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined.

REQUIREMENT OF MICROARRAY EXPERIMENT a) The chip itself with its special surface.

b) The device for producing microarrayas by spotting the nucleic acids onto the chip or for their in situ synthesis.

c) A fluidic system for hybridization of labelled probes to target DNA

d)

A scanner to read the hibridization signals on the chip.

e) Sophisticated software programs to quantify and interpret the results

FABRICATION OF MICROARRAY

Sourcing DNA /clones: What to spot ?

The first step in creating a microarray is to find the source of genes or DNA that will be arrayed on to a glass slide.

a) One can use publicly available clones such as those from the IMAGE consortium or Research Genetics where relatively large number of clones can be obtained relatively inexpensively.

b) Generates clones in house which may be time consuming and expensive.

Preparation and purification of DNA

Microarray can be made by printing plasmids, or most commonly, a section of the plasmid. The relevant part of the plasmid DNA are amplified by PCR, product purity and amount is checked, and then spotted down in concentrated form. Usually a 96 well format is used for this purpose. PCR product should be of similar concentration (approximately 500ng/ul) and sizes (0.2-1.2 kb)

Selection of controls As with any experimental system, the inclusion of relevant controls is essential for the meaningful interpretation of data downstream. Some researcher use housekeeping genes which are expressed constitutively and whose level of expression is thought to be stable.

Deposition of DNA

Deposition of the PCR product are done on glass, nylon or other supports using a robot on Poly-L-Lysine coated glass slides. Typically, 0.5-10 nl of DNA is deposited in a spot 100-150 um in diameter and a distance of 200-250 um from neighboring spots.

Spotting is done in humidity controlled chamber because humidity determines the rate of evaporation of water from the arrayed spots. Rapidly dried spots may be uneven, with most of the DNA in the centr whereas slow drying might result in creeping, and spot spreading. Spo should be perfect circular shape with an even density of DNA. Post deposition processing

After printing, the slides are dried for 24 hours at room temperature. The deposited DNA is then immobilized, usually by UV irradiation

EXPERIMENTS USING MICROARRAY 1. RNA preparation:

High quality total RNA are extracted by guanidium isothiocyanate method and mRNA purified by oligo dT cellulose chromatography. Quality and quantity of RNA should be checked properly (Bioanalyzer)

2. Sample labelling:
A single round of reverse transcription reaction is used to generate a labelled cDNA probe from the sample. Usually 2-5 ug of mRNA is used for labelling with Fluorophores.

Dual label hybridization is a technique often used to compensate for differences in spotted genes. Two samples (normal and experimental are labeled with paired fluorophores (Cy3 and Cy5) that are competitively co-hybridized to the same arary..

3. Hybridization of sample to microarray

Glass microarrays are hybriidized by spotting a small volume of sample (20-25 ul, prepared in hybridization solution) on the microaray and then carefully dropping a coverslip onto it to spread the solution over the entire slide and eliminating the air. The slides are then placed in a humidified (to prevent evaporation) hybridization chamber at 42C (20-25C below the Tm ) for 12-16 hours.

4. Post hybridization processing:

After hybridization, the microarray are subjected to a series of washes to remove unbound, labeled probe and non-specifically bound sequences. During washing stringent conditions are applied by increasing the temperature or lowering the ionic strength of the washing buffer.

5. Image capture :

After hybridization, a digital image of the array is made by a scanner. Most scanner are equipped with confocal laser microscope and a CCD camera.

In practice, the combination of 532 nm (green) and 633nm (red) lasers is used most frequently with Cy3 and Cy5 fluorochromes.

A typical microarray slide after scanning

6. Image Analysis and Data handling:

Once a digital image of the array is obtained, it must be analyzed. The first step in the analysis of data is log transformation of the data. This is because the value from instruments are often biased to small values, with few high values. For each spot, the red to green fluorescence ratio is calculated.

The second step is normalization of data. Rather than obtaining absolute values a ratio of test to control is obtained.

Gene expression changes are often expressed as a fold difference, either greater than two fold or less than 0.5 fold

With respect to handling and analyzing these large amount of data various bioinformatics tools have been introduced and used.

Steps in the design and implementation of a DNA microarray experiment

2) Chip fabrication (Putting probes on the chip)

1) Probe (cDNA/oligo with known identity)

3) Target (fluorecently labeled sample)

4) Assay

5) Readout

6) Informatics

Small oligos, cDNAs, chromosome , ... (whole organism on a chip?)

Photolithogr aphy, RNA, pipette, (mRNA==>) Hybridizatio Fluorescence drop-touch, n,... , piezoelectric cDNA (ink-jet), electric, ...

Image processing, bioinformati cs, data mining and visualization

APPLICATION OD DNA MICROARRAY I. Changes in Gene Expression Levels

The immobilized DNA is cDNA derived from the mRNA of known genes, and the control and sample DNA hybridized to the chip is cDNA derived from the mRNA of normal and diseased tissue, respectively. If a gene is overexpressed in a certain disease state, then more sample cDNA, as compared to control cDNA, will hybridize to the spot representing that expressed gene. In turn, the spot will fluoresce red with greater intensity than it will fluoresce green. Once researchers have characterized the expression patterns of various genes involved in many diseases, cDNA derived from diseased tissue from any individual can be hybridized to determine whether the expression pattern of the gene from the individual matches the expression pattern of a known disease. If this is the case, treatment appropriate for that disease can be initiated. Expression chips can be used to develop new drugs. For instance, if a certain gene is overexpressed in a particular form of cancer, researchers can use expression chips to see if a new drug will reduce overexpression and force the cancer into

II. Genomic Gains and Losses

DNA repair genes are thought to be the body's frontline defense against mutations and, as such, play a major role in cancer. Mutations within these genes often manifest themselves as lost or broken chromosomes. Researchers use a technique called microarray Comparative Genomic Hybridization (CGH) to look for genomic gains and losses or for a change in the number of copies of a particular gene involved in a disease state. In microarray CGH, large pieces of genomic DNA serve as the target DNA, and each spot of target DNA in the array has a known chromosomal location. The hybridization mixture will contain fluorescently labeled genomic DNA harvested from both normal (control) and diseased (sample) tissue. If the number of copies of a particular target gene has increased, a large amount of sample DNA will hybridize to those spots on the microarray that represent the gene involved in that disease, whereas comparatively small amounts of control DNA will hybridize to those same spots. As a result, those spots containing the disease gene will fluoresce red with greater intensity than they will fluoresce green, indicating that the number of copies of the gene involved in

III. Mutations in DNA

Microarrays are used to detect mutations or polymorphisms in a gene sequence. Here, the target, or immobilized DNA, is usually that of a single gene and the target sequence placed on any given spot within the array will differ from that of other spots in the same microarray, sometimes by only one or a few specific nucleotides called a Single Nucleotide Polymorphism, or SNP, a small genetic change or variation that can occur within a person's DNA sequence. Another difference in mutation microarray analysis, as compared to expression or CGH microarrays, is that this type of experiment only requires genomic DNA derived from a normal sample for use in the hybridization mixture. Once researchers have established that a SNP pattern is associated with a particular disease, they can use SNP microarray technology to test an individual for that disease expression pattern to determine whether he or she is susceptible to (at risk of developing) that disease. When genomic DNA from an individual is hybridized to an array loaded with various SNPs, the sample DNA will hybridize with greater frequency only to specific SNPs associated with that person. Those spots on the microarray will then fluoresce with greater intensity, demonstrating that the individual being tested may have, or is at risk for developing, that disease.

In Brief: Microarray Applications

Microarray type

Application

CGH

Tumor classification, risk assessment, and prognosis prediction

Drug development, drug response, and therapy Expression analysis development Mutation/Polymor phism analysis Drug development, therapy development, and tracking disease progression

Microarray Data Management

Considering the amount of data that can potentially be generated using a single microarray chip, many new challenges are presented in terms of data tracking and analysis.

To support the public use and dissemination of gene expression data, NCBI has launched the Gene Expression Omnibus, or GEO.

GEO represents NCBI's effort to build an expression data repository and online resource for the storage and retrieval of gene expression data from any organism or artificial source

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