26-07-2012, 04:15 PM
DNA Microarray
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INTRODUCTION
Microarray technology makes use of the sequence resources created by current genome projects and other sequencing efforts to identify the genes, which are expressed in a particular cell type or an organism .Measuring gene expression levels in variable conditions provides biologists with a better understanding of gene functions, and has wide applications in life sciences. For example, microarrays allow comparison of gene expression between normal and cancerous cells. The technology has been referred by various names: DNA microarrays, DNA arrays, DNA chips, and gene chips.
A microarray is typically a glass slide, onto which DNA molecules are attached at fixed locations, i.e. spots, each related to a single gene. Most of the microarray experiments compare gene-expression from two samples, one called target (or experimental) and the other is called control. The two samples are labeled by synthesizing single stranded cDNAs that are complementary to the extracted mRNA. A typical microarray experiment is depicted . The spots are either printed on the microarrays by a robot, or synthesized by photolithography or ink-jet printing. After the target genes are generated and laid out on the chip surface at defined positions, the cDNA extracted from two samples, labeled with fluorescence dyes, is hybridized to the chip. The result of the binding of cDNA is detected by fluorescence by laser excitation.
In order to obtain the intensity values, the microarray image is processed so that each gene in the microarray is identified as an individual spot, and the intensity of the signal and its surrounding areas are calculated.
The ratio between the signals in the two channels (dyes) is then calculated for each spot.
The result of a microarray experiment is represented as a vector, each element being a spot.
The picture shows a portion of a microarray chip, each spot standing for a gene. The color of each spot reflects the relative abundance of the two fluorescence intensities.
The raw data produced from microarray experiments are called the hybridized microarray images. To obtain information about gene expression levels, these images have to be analyzed: each spot on the array is identified, then its intensity measured and compared to the background. This process is called image quantization. These data can help biologists gain insights into underlying biological processes, only if they are carefully extracted and stored in databases, where they can subsequently be retrieved and analyzed. The overwhelming use of bioinformatics tools for microarray analysis is a significant achievement in biology, because no other technology has used such sophisticated tools, has combined expertise from many different disciplines, and has provided such detailed information about bio sequences.
Although microarrays are a new emerging technology, they have already been widely adopted,
and many users are now going beyond exploratory studies. Microarrays are being exploited in human diseases, drug discovery, and genetic screening and diagnostics . The most promising
commercial application of microarrays is their potential use in clinical diagnostics. Its potential
application goes from drug discovery to gene-based diagnostics, and gene-based treatments. The
most appropriate treatments can be reached by studying the dynamics of gene expressions over time, among tissues, and disease status. In addition, microarrays have a huge potential impact in the areas of preventative medicine, ability to diagnose accurately the disease, and design and screen the drugs that can be used to treat certain disease states.
What Exactly Is a DNA Microarray?
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 With the aid of a computer, the amount of mRNA bound to the spots on the microarray is precisely measured, generating a profile of gene expression in the cell. 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.
TYPES OF ARRAYS
There are predominantly three kinds of microarray technologies in widespread use among most laboratories: spotted microarrays consisting of presynthesized oligos or PCR products robotically deposited onto a surface, AffymetrixGeneChips composed of relatively short oligonucleotides synthesized on a chip surface, and other in situ synthesis platforms such as arrays made by Agilent and NimbleGen. Although each technology effectively serves as a genomic readout, each has unique characteristics that offer advantages or disadvantages in a given context. Parallel forms of measuring DNA and RNA will continue to change and evolve; however, these three platforms are currently the most ubiquitous.
2.1 Spotted Microarrays
Spotted microarrays were the first widely available array platform and continue to enjoy broad use. Originating in the laboratory of Pat Brown, they consist of glass microscope slides onto which libraries of PCR products or long oligonucleotides are printed using a robot equipped with nibs capable of wicking up DNA from micro titer plates and depositing it onto the glass surface with micron precision [13,15]. Since their inception, demand for microarrays has exceeded availability. Because the Brown laboratory expended effort in every aspect of distributing the technology, including plans to build the robot and all protocols required for array manufacture
and use, many academic laboratories invest resources into producing these arrays locally. This includes building or purchasing a robot, as well as performing PCR or oligo design and synthesis to create probes for spotting onto glass. The basic principle by which the arrays function is fairly simple, and all the reagents required are available to most researchers with some initial investment. However, apart from praising the benefits of putting technology into the hands of researchers, the reason for highlighting this aspect of spotted arrays is to point out the nonuniform nature of spotted microarrays. Because there is not one manufacturer, one source of materials, or a uniform method of production, variability exists among batches of microarrays
and must be considered when planning experiments or when comparing experiments from different array sources.
Spotted microarrays are primarily a comparative technology. They are used to examine relative concentrations of targets between two samples. Complex samples to be compared are labeled with uniquely colored fluorescent tags before being mixed together and allowed to compete for hybridization to the microarray spots. In this way, differences between the samples are observed on a per spot basis because the fractional occupancy of the spot hybridized by each sample reflects the relative concentration of that gene or target in the original complex mixture. Thus, for any probe on the microarray, one gets a readout of the relative concentrations of the target between the two input samples. For this reason, spotted microarrays are often called two-color or two-sample arrays.
Affymetrix GeneChips
Affymetrix GeneChips are the most ubiquitous and long-standing commercial array platform in use. The arrays consist of 25-mer oligonucleotides synthesized in situ on the surface of a glass chip. Aphotolithography mask, similar to that used to construct semiconductor chips, is used to control light-directed DNA synthesis chemistry such that oligo sequences are built up one nucleotide at a time at defined locations on a solid substrate or glass chip [18,19]. Current chips contain 6.5 million unique probes in an area of 1.28 cm2. The highly precise nature of the lithographic method allows the construction of compact matrices of square patches of probes.
Instead of using a single sequence to probe expression of each gene, as would be common for a spotted array, Affymetrix employs a set of probes to measure expression of a gene. Probe sets contain two types of probes to measure the gene of interest, perfect match (PM) and mismatch (MM) probes. Perfect match probes are chosen to match the gene exactly and are designed against an exemplar sequence representing the gene. Although each probe is unique, probes may occasionally overlap. Mismatch probes are identical to the perfect match probes except that they contain a single base mismatch in the center of the probe. A single mismatch in a short sequence such as a 25-mer is very disruptive to hybridization. The purpose of the mismatch probe is to serve as a negative control for background hybridization. A typical probe set contains 11 perfect match probes and 11 mismatch probes. The positioning of probes for a single gene on the array is chosen by a random process to protect against local hybridization artifacts that could otherwise affect all the probes for a gene if they were clustered together. As most spotted arrays use only one probe per gene, local hybridization artifacts can be a problem.
Affymetrix GeneChips are single sample microarrays (also known as one color or one channel). These arrays measure the relative abundance of every gene in a single sample. In this way, one can examine whether one gene is expressed at a higher or lower level than some other gene in the same sample. If samples are to be compared, a separate chip must be performed for each sample, and the data adjusted by scaling or normalization before comparison.
Other In Situ Synthesis Platforms
Apart from Affymetrix, two alternative in situ synthesis methods exist by which oligonucleotides are built up one nucleotide at a time in successive steps to create probes of length 25–60 nucleotides long [108]. These methods are almost exclusively commercial and different companies take different approaches. Although Affymetrix uses a mask-based photolithographic process to control light-directed DNA synthesis, an alternative method employed by NimbleGen makes use of small rotating mirrors to control light and accomplish a similar task [22]. This approach is called Maskless Photolithography, and uses technology developed by Texas Instruments for projection televisions in which arrays of digitally controlled micromirrors can be used to direct light. In combination with light activated chemistry, light of the appropriate intensity and wavelength can be actuated in patterns required to build up any series of nucleotides into an oligonucleotide on a solid surface [23,24].