Over the past decade, more than 30 organisms have had their genomes completely sequenced, including our own genome. But all of this information does not tell us what all the genes do, how cells work, how cells form organisms, how we age, what goes wrong in disease or how to develop a drug. This is where functional genomics comes into play. The purpose of genomics is to understand biology, not simply to identify the component parts.
DNA Microarrays
Among the most powerful tools for genomics are high-density arrays of oligonucleotides or parts of cDNAs. Nucleic acid arrays work by hybridization of labelled RNA or DNA in solution to DNA molecules attached at specific locations on a surface. Although it is possible to synthesize or deposit DNA fragments of unknown sequence, the most common implementation is to design arrays based on specific sequence information.
DNA microarrays follows this general procedure:
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the chip itself with a special surface. Using technology from the semiconductor industry, manufacture begins with a 5 inch surface quartz wafer which is washed to insure uniform hydroxylation.
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the device for producing microarrays by spotting the nucleic acids (probes) onto the chip.
High-density Microarray (HDMs): In one highly used method, probe synthesis occurs in parallel resulting in the addition of A, C, T or G to multiple growing chains simultaneously. To define which oligonucleotide chains will receive a nucleotide in each step, photolithographic masks, carrying 18 to 20 square micron windows that correspond to the dimensions of individual features, are placed over the coated wafer. When ultraviolet light is shone over the mask in the first step of synthesis, the exposed linkers become deprotected and are available for nucleotide coupling. Critical to this step is the precise alignment of the mask with the wafer before each synthesis step. To ensure that this critical step is accurately completed, chrome marks on the wafer and on the mask are perfectly aligned. For more information click here
In these arrays, the so called oligonucleotide type microarrays, each gene is represented by multiple probe pairs with a bias toward the 3`end of the gene. Each probe pair consists of a perfect match and a mismatch oligonucleotide (that is, on average, 20 base pairs in length). The mismatch oligonucleotide contains a single base pair mismatch in the center of the probe. The mismatch is designed to measure background. The software subtracts the hybridization intensities of the mistmatch oligos from those of the perfect match oligos to determine the absolute or specific intensity value for each oligo set. Specifically, total or PolyA-selected RNA is used to create double stranded cDNA with a T7 RNA polymerase site incorporated. An in vitro transcription is run on this DNA, with biotinylated nucleotides included in the mix. This biotinylated aRNA is the target that is hybridized to the GeneChip®. During the washing and staining procedure streptavidin-phycoerythrin is used to make the biotin labeled aRNA flourescent. The chip is then scanned to capture an image of the fluorescence of each feature. The Affymetrix software measures the intensity of the signal from each perfect match probe, compares it to the signal for the mismatch probe, does this across all the pairs in a probe set for a given gene and, using a statistical algorithm, comes up with a call as to whether or not the gene is present in the original sample and a measure of the expression level if present.
Spotted Microarrays (SMs)
In another methods, spotted arrays can be made from oligonucleotides as short as 30 mers, or clones as long as 2 kb. The DNA is put into a PCR plate and loaded onto the robot. The robot maneuvers pins to pick up the DNA solution and deposit it onto glass slides in precise predefined locations. To compare the gene expression between two tissue samples, cDNA from each sample is prepared using a separate florescent dye. The two samples are then mixed together and put on the microarray. Each spot on the microarray will indiscriminantly hybridize the corresponding cDNA from each sample. If one sample contained more mRNA of a certain gene than the other sample, then the microarray spot for that gene will emit a higher florescent signal when viewed under the appropriate light source. Using a high-resolution laser scanner and sophisticated software programs, the intensity in each channel for each spots is analyzed, revealing the difference in the expression level for that gene between the two samples. Numbers are relative and expressed as the ratio of expression level in the control sample vs an experimental sample.
Bead Arrays (BAs)
BAs do not involve planar substrates, but are comprised of an addressable population of microscopic polymer beads that contain precise amounts of up to four different fluorophores. Each type of bead has a characteristic DNA target suface coating where the identify of each tpe of bead is then determined optically by measuring the relative fluorescence from each fluorophore.
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labeled cDNA or cRNA targets derived from the mRNA of an experimental sample are hybridized to nucleic acid probes attached to the solid support. By monitoring the amount of label associated with each DNA location, it is possible to infer the abundance of each mRNA species represented. Although hybridization has been used for decades to detect and quantify nucleic acids, the combination of the miniaturization of the technology and the large and growing amounts of sequence information, have enormously expanded the scale at which gene expression can be studied
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a scanner is used to read the chips;
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oftware programs to quantify and interpret the results.
Applications and advantages of DNA microarrays include the following:
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Gene expression profiling (mRNA abundance): The transcription of genomic DNA to produce mRNA is the first step in the process of protein synthesis, and differences in gene expression are responsible for both morphological and phenotypic differences as well as indicative of cellular responses to environmental stimuli and pertubations. In terms of understanding the function of genes, knowing when, where and to what extent a gene is expressed is central to understanding the activity and biological roles of its encoded protein.
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Entire genomes can be probed: It is no longer necessary to guess what the important genes are in advance. Now entire genomes can be included on a DNA microarray. Conventional methods such as northern and western blots or RT-PCR can still be used for measuring gene expression at the mRNA level as a follow up on genes that have been implicated by array results.
Possible disadvantages to DNA microarrays are the following:
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Disparity between the relative expression levels of mRNA and their corresponding proteins.
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Posttranslational protein modification, protein-protein interactions and protein -DNA interactions are not taken into account.
Protein Microarrays
There are 2 general types of proteins microarrays: analytical and functional. Analytical microarrays involve a high-density array of affinity reagents (e.g. antibodies or antigens) that are used for detecting proteins in a complex mixture. Functional protein chips are constructed by immobilizing large numbers of purified proteins on a solid surface and have enormous potential in assaying for a wide range of biochemical activities (e.g., protein-protein, protein-lipid, protein-nucleic acid and enzyme-substrate interactions) as well as drug and drug target identification.
Antibody Microarrays: are arrays of antibodies on a glass slide that bind specific antigens. A lysate is passed over the array and the bound antigen is detected after washing. Detection is usually carried out by using labeled lysates or using a second antibody that recognizes the antigen of interest.
- large range of protein expression: An example of this is where reagents might have high affinity for one protein but low affinity for another. Such reagents will still exhibit detection of the lower affinity protein if it is much more prevalent. Using sandwich assays can increase the specificity, however, but requires that at least 2 high quality antibodies exist for each antigen to be detected.
Possible disadvantages to consider with antibody microarrays are the following:
- requirement of detection antibodies which greatly limits the development of a higher density protein array system because it is very difficult to mix hundred or even thousands of antibodies together for high density protein arrays.
- possibility of cross-reactivity between antigens with an antibody.
Protein Chips: have become instrumental in learning about . These chips are constructed by immobilizing large numbers of purified proteins on a solid surface. Immobilization can be by absorption, covalent cross-linking and affinity
attachment. Applications and advantages of protein chips are the following:
- As one example, 5800 yeast ORFs were fused to GST-HisX6 at their NH2 termini and expressed in yeast using the inducible GAL1 promoter. The yeast expression strains contained individual plasmids in which the ORFs were fused in frame to GST. These fusion proteins were then printed onto glass slides using a commercially available microarrayer and then screened for their ability to interact with proteins and phospholipids. For example, the proteome was probed with a biotenylated protein of interest which was then detected suing streptavidin. (Zhu et al. Science 291:2101-2105,2001).
- Lam (US 5,510240) discloses a method of generating a random library of bio-oligomers such as peptides of a defined size and known composition attached to solid phase supports wherein each solid phase support is attached to a single bio-oligomer species and all possible combinations of monomer subunits of which the bio-oligomers are composed are included in the collection. The method includes the steps of completely coupling a subunit to substantially all sites of a solid support to form a solid phase support/new subunit combination. In the case of a peptide, for example, the coupling of the amino acids may be done by techniques well known in the art such as building a peptide from the carboxyl or C terminal end in which the C-terminal amino acid with its alpha-amino group protected is attached to a solid phase polymer. The protecting roup is then cleaved off and the next amino acid, also protected, is coupled by a peptide bond to the alpha-minao group of the amino acid attached to the solid support. The cycle of deprotection of the prior amino acid and coupling of the additional amino acid is repeated until the peptide is completed. In order to couple an amino acid to the growing synthetic chain, the carobxyl group of the blocked amino acid must be activated through methods known in the art. The biol-oligomer library can then be used to identify bio-oligoers within the library that demonstrate a biological activity of interest.
Although there has been a lot of progress in the development of protein chips, proteins, by their nature, present many challenges for protein chips such as the following:
- lack of suitable amplification methods: Whereas one can use PCR to amplify DNA for DNA chips one must rely on different prokaryotic and eukaryotic expression systems to obtain proteins.
- Attachment to substrate: One challenge here is to make sure that the active site of the protein is not blocked due to the attachment.
- multiple forms of the same gene products due to posttranslational modifications, splice variants, etc.
Manufacturers of protein microarrays: Ciphergen Biacore Perkinelmer
Cell Microarrays
Cell microarrays use a microarray pin transfer device to transfer nanoliter volumes of a gelatin solution containing cDNA clones in expression vectors onto a slide. Transfection reagent and cells are then added, creating a cell microarray. Each array feature is a cell cluster overexpressing a specific cDNA. The array is then removed from media. The procedure is as following:
(1) nonoliter quantities of cDNA containing plasmids are dissolved in an aqueous gelatin solution and printed onto the surface of a glass slide using a robotic microarrayer device.
(2) the printed arrays are then briefly exposed to a lipid transfection reagent, resulting in the formation of lipid-DNA complexes on the surface of the slide.
(3) mammalian cells in medium are added on top of the array in culture dishes, and cells that grow on top of the area where plasmids were printed take up these plasmids and become transfected. The result is a living microarray in which each feature is a cluster of 30-80 cells overexpressing a particular gene product.
(4) to visualize cell microarrays, the array slides can be fixed and a variety of detection assays can be applied, including in situ hybridization, immunofluorescence and autoradiography.