The Basics of DNA Microarrays
The Human Genome Project has created a massive amount of DNA sequence information. To take full advantage of this latest information, scientists have developed new techniques and tools for conducting research. DNA microarrays, which are also called DNA arrays or gene chips, are an example of a tool that uses genome sequence information to analyze the structure and function of tens of thousands of genes at a time.
How Do Arrays Work?
DNA arrays come in many varieties. Whether they are created by scientists or produced commercially by one of several companies, arrays depend on the same basic principle: Complementary sequences of nucleotides stick to, or hybridize to, one another. For example, a DNA molecule with the sequence -A-T-T-G-C- will hybridize to another with the sequence -T-A-A-C-G- to form double-stranded DNA.
For the past 25 years, scientists have been using hybridization as a standard technique to detect specific DNA or RNA sequences. A single-stranded DNA molecule with a known sequence is labeled with a radioactive isotope or fluorescent dye and then used as a probe to detect a fragment of DNA or messenger RNA (mRNA, the molecule that is produced when a gene is turned on or expressed) with the complementary sequence. For example, if a researcher wants to know whether gene A is expressed in a particular tissue, the researcher would make a radio-labeled DNA probe by using a small piece of gene A, isolate mRNA from the tissue of interest, bind the mRNA to a solid medium (such as a nylon filter), and then hybridize the probe to the filter. If gene A is expressed in the tissue, the researchers will see a radioactive signal on the filter. This procedure is known as a Northern blot. Imagine the power of being able to do thousands of these experiments at a time.
DNA microarrays use the same DNA probe detection method but on a much larger scale. Instead of detecting one gene or one mRNA at a time, microarrays allow thousands of specific DNA or RNA sequences to be detected simultaneously on a glass or plastic slide about 1.5 centimeters square (about the size of your thumb). Each microarray is made up of many bits of single-stranded DNA fragments arranged in a grid pattern on the glass or plastic surface. When sample DNA or RNA is applied to the array, any sequences in the sample that find a match will bind to a specific spot on the array. A computer then determines the amount of sample bound to each spot on the microarray.
An Array of Applications
Gene Expression Arrays
DNA arrays are commonly used to study gene expression. In this type of study, mRNA is extracted from a sample (for example, blood cells or tumor tissue), converted to complementary DNA (cDNA), which is easier to work with than RNA, and tagged with a fluorescent label. In a typical microarray experiment, cDNA from one sample (sample A) is labeled with red dye and cDNA from another (sample B) with green dye. The fluorescent red and green cDNA samples are then applied to a microarray that contains DNA fragments corresponding to thousands of genes. (For certain organisms whose genomes have been sequenced, such as the roundworm Caenorhabditis elegans, there are arrays that represent all the genes in the genome.) If a DNA sequence is present both on the array and on one or both samples, the sequences bind, and a fluorescent signal sticks to a specific spot on the array. The signals are picked up using a reader or scanner that consists of lasers, a special microscope, and a camera, which work together to create a digital image of the array. Special computer programs then calculate the red to green fluorescence ratio in each spot. The calculated ratio for each spot on the array reflects the relative expression of a given gene in the two samples. (For example, a red signal indicates that a particular gene is expressed in sample A but not sample B; a green signal that the gene is expressed in sample B but not sample A; and a yellow signal that the gene is expressed at roughly equal levels in both samples. No signal means that the gene is not expressed in either sample.) The result of a gene expression experiment is referred to as a gene expression profile or signature.
Expression arrays can be used to answer basic biology problems. For example, by comparing the expression profile of a cell that is in a resting state to the profile of one that is dividing, scientists can determine which genes are turned on during cell division. Microarrays also have medical applications. For example, by comparing the expression profile of a cancer cell with that of a normal cell, scientists can use microarrays to diagnose different cancers. Better diagnosis can lead to more-informed treatment choices.
Eric Landers group was the first to show that gene expression arrays could be used to distinguish between two types of leukemia, acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In a paper published in 1999 in the journal Science, the scientists used expression results from 6,800 human genes measured on arrays to accurately predict whether a patient had AML or ALL. Until then, doctors distinguished between the two cancers by using a battery of expensive tests that could cost critical time. Since that study, a number of studies have shown similar results with different types of cancers.
DNA Sequence Arrays
Other types of arrays can be used to measure genetic variations among individuals. For example, single nucleotide variations, or SNPs (pronounced snips), which occur throughout the genome, can be detected by using genotyping arrays or SNP chips. These types of arrays carry all the possible variations of one gene or several genes in a grid pattern. A DNA sample is extracted, multiple copies of the gene or genes of interest are generated using polymerase chain reaction (PCR), and the sample is then applied to the chip. The spots that light up correspond to the particular gene variants the individual has.
Like the expression arrays, these types of arrays provide basic information, such as the range of variation in human genomes. But they can also have clinical applications. By looking at several SNPs at once, researchers can identify SNP signatures associated with a specific disease or a response to a drug. Individuals at risk for a particular disease could then be tested for the telltale signature.
A newer technique called microarray comparative genomic hybridization (microarray CGH) has been developed to identify large regions of DNA that are either missing (deletions) or present in more copies (amplifications). Rearrangements in the DNA, such as deletions and amplifications, are often involved in diseases such as cancer. Other types of arrays are able to detect DNA sequences that bind to proteins or sequences that are chemically modified in the genome, such as by methylation or acetylation.
A different but conceptually similar approach is being applied directly to proteins. Scientists have developed microarrays that can be used either to identify and quantify thousands of different proteins at once or to find associations between different kinds of proteins and between proteins and other molecules. These types of arrays are collectively referred to as protein arrays.
Protein arrays that are used to identify proteins typically consist of many antibodies arrayed on a glass or plastic slide. Each antibody can bind to a different target protein. When a mixture of proteinsfor example, in a blood sampleis applied to the array, the proteins recognized by the different antibodies will bind to the array. Bound proteins can be detected either by adding a second antibody tagged with a fluorescent molecule or by chemically labeling all the proteins in the blood sample before adding the sample to the array. Each bound protein can therefore be detected as a signal on the array, and the intensity of the signal roughly represents the amount of protein in the blood sample. This type of array, like a gene expression array, can be used to generate signatures for different cell types and tissues.
Another type of protein microarray is used to glean insights into the function of different proteins by looking at the molecules they bind to. For this application, the proteins themselves, rather than their antibodies, are arrayed on a slide. Stuart Schreibers group was one of the first to show that more than 10,000 different proteins could be stuck to a single glass microscope slide and still retain their biological activity. In a typical experiment, thousands of proteins that are found in a cell are bound to an array. Then a particular type of moleculefor example, a fat molecule (or lipid)is fluorescently labeled and applied to the protein array. The spots on the array that light up with a fluorescent signal correspond to proteins that associate with lipids. This knowledge provides important insights into the function of many proteins at once.
Protein binding information can also be obtained by using a small molecule microarray, in which thousands of different small synthetic molecules are arrayed on a slide. An advantage of this type of array is that small molecules tend to be more stable and rugged, which simplifies storage and handling requirements and makes the process suitable for mass production. By letting a single protein species react to this type of array, one can identify different small molecules that bind to proteins. Some of these small molecules may be candidates for a reagent that interferes with the functions of a protein, or they may even lead to new drug discovery.
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