OLIGONUCLEOTIDE(DNA) MICROARRAY CHIP

INTRODUCTION

DNA Microarray Technology

Although all of the cells in the human body contain the same genetic material, the same genes are not active in all of those cells ( Differentially expressed). Studying which genes are active and which are inactive in different kinds of cells helps scientists understand more about how these cells function and about what happens when the genes in a cell dont function properly. In the past scientists have only been able to conduct such genetic analyses on a few genes at once. With the development of DNA microarray technology, however, scientists can now examine thousands of genes at the same time, an
advance that will help them determine the complex relationships betweenindividual genes. DNA Microarrays are small, solid supports onto which the sequences from thousands of different genes are immobilized, or attached, at fixed locations. The supports themselves are usually glass microscope slides, the size of two side-by-side pinky fingers, but can also be silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support. The American Heritage Dictionary defines "array" as "to place in an orderly arrangement". It is important that the gene sequences in a microarray are attached to their support in an orderly or fixed way, because a researcher uses the location of each spot in the array to identify a particular gene sequence. The spots themselves can be DNA, cDNA, or oligonucleotides. Microarrays are simply ordered sets of DNA molecules of known sequence. Usually rectangular, they can consist of a few hundred to hundreds of thousands of sets. Each individual feature goes on the array at precisely defined location on the substrate. The identity of the DNA molecule fixed to each feature never changes. Scientists use that fact in calculating their experimental results. Microarray analysis permits scientists to detect thousands of genes in a small sample simultaneously and to analyze the expression of those genes. As a result, it promises to enable biotechnology and pharmaceutical companies to identify drug targets - the proteins with which drugs actually interact.

Genomics refers to the comprehensive study of genes and their function. Recent advances in bioinformatics and high-throughput technologies such as microarray analysis are bringing about a revolution in our understanding of the molecular mechanisms underlying normal and dysfunctional biological processes. Microarray studies and other genomic techniques are also stimulating the discovery of new targets for the treatment of disease which is aiding drug development, immunotherapeutics and gene therapy. Since it can also help identify individuals with similar biological patterns, microarray analysis can assist drug companies in choosing the most appropriate candidates for participating in clinical trials of new drugs. In the future, this emerging technology has the potential to help medical professionals select the most effective drugs, or those with the fewest side effects, for individual patients.

METHODOLOGY

Gene expression profiling or microarray analysis has enabled the measurement of thousands of genes in a single RNA sample. There are a variety of microarray platforms that have been developed to accomplish this and the basic idea for each is simple: 1. a glass slide or membrane is spotted or "arrayed" with DNA fragments or oligonucleotides that represent specific gene coding regions. 2. Purified RNA is then fluorescently- or radioactively labeled and hybridized to the slide/membrane. In some cases, hybridization is done simultaneously with reference RNA to facilitate comparison of data across multiple experiments. 3. After thorough washing, the raw data is obtained by laser scanning or autoradiographic imaging (Figure 1). At this point, the data may then be entered into a database and analyzed by a number of statistical methods.

Figure 1: Principle of DNA microarray analysis The targets for microarray analysis are two pools of fluorescently labelled cDNAs derived from mRNA of control and experiment cells.

A DNA Microarray Experiment

1. Prepare your DNA chip using your chosen target DNAs. 2. Generate a hybridization solution containing a mixture of fluorescently labeled cDNAs. 3. Incubate your hybridization mixture containing fluorescently labeled cDNAs with your DNA chip.

4. Detect bound cDNA using laser technology and store data in a computer.

5. Analyze data using computational methods.

How does microarray work?

 Microarray is based on a database of over 40,000 fragments of genes called expressed sequence tags (ESTs) in case of Humans. Hundreds or thousands of these ESTs ( cDNA fragments) are arranged on a single microscope slide (DNA microchip) by a robot. The DNA microchip consists of a small glass plate encased in plastic. On the surface, each chip contains synthetic single stranded DNA sequences identical to a normal gene. Isolation of RNA from different tissues where the gene is suppose to be differentially expressed or target tissues. Separation of m-RMA from whole RMA family by passing the sample from oligo-dT column. Construction of cDNA library as a representative of genes in question. (DNA is more stable molecule in comparison to RNA). Next representative of mRNA i.e cDNA from a particular cells are labeled with fluorescent tags and allowed to hybridize, or bind, to the ESTs/ cDNA probes on the slide. After a scanner measures the fluorescence of each sample on the slide, scientists can determine how active the genes represented by the ESTs are in the cell. Strong fluorescence indicates that many of the cells messengers hybridized to the EST and, therefore, that the gene is very active in the cell. Conversely, no fluorescence indicates that none of the messenger molecules hybridized to the EST and that the gene is inactive in the cell.

Statistical analysis The analysis of DNA microarrays poses a large number of statistical problems, including the normalization of the data. There are dozens of proposed normalization methods in the published literature; a sound conservative approach is to try a number of popular normalization methods and compare the conclusions reached: how sensitive are the main conclusions to the method chosen? From a hypothesis-testing perspective, the large number of genes present on a single array means that the experimenter must take into account a multiple testing problem: even if each gene is extremely unlikely to randomly yield a result of interest, the combination of all the genes is likely to show at least one or a few occurrences of this result which are false positives. A basic difference between microarray data analysis and much traditional biomedical research is the dimensionality of the data. A large clinical study might collect, say, 100 data items per patient for thousands of patients. A medium-size microarray study will obtain many thousands of numbers per sample for perhaps a hundred samples. Many analysis techniques treat each sample as a single point in a space with thousands of dimensions, then attempt by various techniques to reduce the dimensionality of the data to something humans can visualize. Relation between probe and gene The relation between a probe and the mRNA that it is expected to detect is problematic. On the one hand, some mRNAs may cross-hybridize probes in the array that are supposed to detect another mRNA. On the

other hand, probes that are designed to detect the mRNA of a particular gene may be relying on genomic EST information that is incorrectly associated with that gene.

TYPES OF MICROARRAY

Protein microarray

A protein microarray is a piece of glass on which different molecules of protein have been affixed at separate locations in an ordered manner thus forming a microscopic array. These are used to identify protein-protein interactions, to identify the substrates of protein kinases, or to identify the targets of biologically active small molecules. The most common protein microarray is the antibody microarray, where antibodies are spotted onto the protein chip and are used as capture molecules to detect proteins from cell lysate solutions.

Applications
Protein microarrays (also biochip, proteinchip) are measurement devices used in biomedical applications to determine the presence and/or amount (referred to as quantitation) of proteins in biological samples, e.g. blood. They have the potential to be an important tool for proteomics research. Usually a multitude of different capture agents, most frequently monoclonal antibodies, are deposited on a chip surface (glass or silicon) in a miniature array. This format is often also referred to as a microarray (a more general term for chip based biological measurement devices).

Antibody microarray
An antibody microarray is a specific form of protein microarrays, a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose of detecting antigens. Antibody microarray is often used for detecting protein expressions from cell lysates in general research and special biomarkers from serum or urine for diagnostic applications.

Tissue microarray
consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow simultaneous histological analysis. The major limitations in molecular clinical analysis of tissues include the cumbersome nature of procedures, limited availability of diagnostic reagents and limited patient sample size. The technique of tissue microarray was developed to address these issues. Multi-tissue blocks were first introduced by H. Battifora in 1986 with his so called multitumor (sausage) tissue block" and modified in 1990 with its improvement, "the checkerboard tissue block" . In 1998 J. Kononen and collaborators developed the current technique, which uses a novel sampling approach to produce tissues of regular size and shape that can be more densely and precisely arrayed.

a Tissue MicroArray Section In the tissue microarray technique, a hollow needle is used to remove tissue cores as small as 0.6 mm in diameter from regions of interest in paraffin embedded tissues such as clinical biopsies or tumor samples. These tissue cores are then inserted in a recipient paraffin block in a precisely spaced, array pattern. Sections from this block are cut using a microtome, mounted on a microscope slide and then analyzed by any method of standard histological analysis. Each microarray block can be cut into 100 500 sections, which can be subjected to independent tests. Tests commonly employed in tissue microarray include immunohistochemistry, and fluorescent in situ hybridization. Tissue microarrays are particularly useful in analysis of cancer samples.