The Basics of DNA Micro Arrays

8/14/2019 The Basics of DNA Micro Arrays

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The Basics of DNA Microarrays

The Human Genome Project has created a massive amount of DNA sequence information. Totake full advantage of this latest information, scientists have developed new techniques andtools 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 producedcommercially 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 todetect specific DNA or RNA sequences. A single-stranded DNA molecule with a known sequenceis labeled with a radioactive isotope or fluorescent dye and then used as a probe to detect afragment of DNA or messenger RNA (mRNA, the molecule that is produced when a gene isturned on or expressed) with the complementary sequence. For example, if a researcherwants to know whether gene A is expressed in a particular tissue, the researcher would make aradio-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 theprobe to the filter. If gene A is expressed in the tissue, the researchers will see a radioactivesignal on the filter. This procedure is known as a Northern blot. Imagine the power of beingable to do thousands of these experiments at a time.

DNA microarrays use the same DNA probe detection method but on a much larger scale. Insteadof detecting one gene or one mRNA at a time, microarrays allow thousands of specific DNA orRNA sequences to be detected simultaneously on a glass or plastic slide about 1.5 centimeterssquare (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 sampleDNA or RNA is applied to the array, any sequences in the sample that find a match will bind to aspecific spot on the array. A computer then determines the amount of sample bound to eachspot on the microarray.


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An Array of Applications

Gene Expression Arrays

DNA arrays are commonly used to study gene expression. In this type of study, mRNA isextracted from a sample (for example, blood cells or tumor tissue), converted tocomplementary DNA (cDNA), which is easier to work with than RNA, and tagged with afluorescent label. In a typical microarray experiment, cDNA from one sample (sample A) islabeled with red dye and cDNA from another (sample B) with green dye. The fluorescent redand green cDNA samples are then applied to a microarray that contains DNA fragmentscorresponding to thousands of genes. (For certain organisms whose genomes have beensequenced, such as the roundworm Caenorhabditis elegans, there are arrays that represent allthe genes in the genome.) If a DNA sequence is present both on the array and on one or bothsamples, the sequences bind, and a fluorescent signal sticks to a specific spot on the array. Thesignals are picked up using a reader or scanner that consists of lasers, a specialmicroscope, and a camera, which work together to create a digital image of the array. Specialcomputer programs then calculate the red to green fluorescence ratio in each spot. Thecalculated ratio for each spot on the array reflects the relative expression of a given gene inthe two samples. (For example, a red signal indicates that a particular gene is expressed insample A but not sample B; a green signal that the gene is expressed in sample B but notsample A; and a yellow signal that the gene is expressed at roughly equal levels in bothsamples. 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 comparingthe 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 havemedical applications. For example, by comparing the expression profile of a cancer cell withthat of a normal cell, scientists can use microarrays to diagnose different cancers. Betterdiagnosis can lead to more-informed treatment choices.


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Eric Landers group was the first to show that gene expression arrays could be used todistinguish between two types of leukemia, acute lymphoblastic leukemia (ALL) and acutemyeloid leukemia (AML). In a paper published in 1999 in the journal Science, the scientists usedexpression results from 6,800 human genes measured on arrays to accurately predict whether apatient had AML or ALL. Until then, doctors distinguished between the two cancers by using abattery 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. Forexample, single nucleotide variations, or SNPs (pronounced snips), which occur throughoutthe genome, can be detected by using genotyping arrays or SNP chips. These types of arrayscarry all the possible variations of one gene or several genes in a grid pattern. A DNA sample isextracted, multiple copies of the gene or genes of interest are generated using polymerasechain reaction (PCR), and the sample is then applied to the chip. The spots that light upcorrespond 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 atseveral SNPs at once, researchers can identify SNP signatures associated with a specificdisease or a response to a drug. Individuals at risk for a particular disease could then be testedfor the telltale signature.

A newer technique called microarray comparative genomic hybridization (microarray CGH) hasbeen developed to identify large regions of DNA that are either missing (deletions) or presentin more copies (amplifications). Rearrangements in the DNA, such as deletions andamplifications, are often involved in diseases such as cancer. Other types of arrays are able todetect DNA sequences that bind to proteins or sequences that are chemically modified in thegenome, such as by methylation or acetylation.

Protein Arrays

A different but conceptually similar approach is being applied directly to proteins. Scientistshave 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 andbetween proteins and other molecules. These types of arrays are collectively referred to asprotein arrays.

Protein arrays that are used to identify proteins typically consist of many antibodies arrayed ona 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 bythe different antibodies will bind to the array. Bound proteins can be detected either by addinga second antibody tagged with a fluorescent molecule or by chemically labeling all the proteinsin the blood sample before adding the sample to the array. Each bound protein can thereforebe detected as a signal on the array, and the intensity of the signal roughly represents theamount of protein in the blood sample. This type of array, like a gene expression array, can beused to generate signatures for different cell types and tissues.

Another type of protein microarray is used to glean insights into the function of differentproteins by looking at the molecules they bind to. For this application, the proteinsthemselves, rather than their antibodies, are arrayed on a slide. Stuart Schreibers group wasone of the first to show that more than 10,000 different proteins could be stuck to a singleglass microscope slide and still retain their biological activity. In a typical experiment,


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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 theprotein array. The spots on the array that light up with a fluorescent signal correspond toproteins that associate with lipids. This knowledge provides important insights into the functionof many proteins at once.

Protein binding information can also be obtained by using a small molecule microarray, in whichthousands of different small synthetic molecules are arrayed on a slide. An advantage of thistype of array is that small molecules tend to be more stable and rugged, which simplifiesstorage and handling requirements and makes the process suitable for mass production. Byletting a single protein species react to this type of array, one can identify different smallmolecules that bind to proteins. Some of these small molecules may be candidates for areagent that interferes with the functions of a protein, or they may even lead to new drugdiscovery.


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Any chromosome other than a sex chromosome. Humans have 22 pairs of autosomes.


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Cytogenic Map

The visual appearance of a chromosome when stained and examined under a microscope.Particularly important are visually distinct regions, called light and dark bands, which give eachof the chromosomes a unique appearance. This feature allows a person's chromosomes to bestudied in a clinical test known as a karyotype, which allows scientists to look for chromosomal

alterations.


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DNA ReplicationThe process by which the DNA double helix unwinds and makes an exact copy of itself.


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EXONThe region of a gene that contains the code for producing the gene's protein. Each exon codesfor a specific portion of the complete protein. In some species (including humans), a gene'sexons are separated by long regions of DNA (called introns or sometimes "junk DNA") that haveno apparent function.

fluorescence in situ hybridization (FISH)

A process which vividly paints chromosomes or portions of chromosomes with fluorescentmolecules. This technique is useful for identifying chromosomal abnormalities and genemapping.


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MarkerAlso known as a genetic marker, a segment of DNA with an identifiable physical location on achromosome whose inheritance can be followed. A marker can be a gene, or it can be somesection of DNA with no known function. Because DNA segments that lie near each other on achromosome tend to be inherited together, markers are often used as indirect ways of trackingthe inheritance pattern of genes that have not yet been identified, but whose approximate

locations are known.


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Noncoding DNA

The strand of DNA that does not carry the information necessary to make a protein. The non-coding strand is the mirror image of the coding strand and is also known as the antisensestrand.


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Polymerase Chain Reaction - PCR A fast, inexpensive technique for making an unlimited number of copies of any piece of DNA.Sometimes called "molecular photocopying," PCR has had an immense impact on biology andmedicine, especially genetic research.

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The Basics of DNA Micro Arrays