Probes, microarray

For proteins microarrays, most protocols fink fluorescent dyes to proteins in a covalent bond via Lys and Arg residues. Fluorescein isothiocyanate (FITC) contains a reactive 

The specificity and affinity of interactions between target molecules bound to the microarray substrate and probe molecules in solution largely determine the quality of microarray assays. The complementary base hybridization is the most efficient and reproducible target-probe interactions used in DNA microarray analysis. 

Microarray analysis provides a technology platform for massive, parallel analysis of protein-protein interactions (MacBeath and Schrieber, 2000 Joos et al 2002). The difficulty with protein microarray is that proteins do not behave as uniformly as nucleic acids. Protein function is dependent on a precise and fragile 3D structure that may be difficult to maintain in a microarray format A practical challenge posed by proteins derives from their relatively delicate tertiary stracture, which is susceptible to unfolding during microarray printing. In addition, the efficiency and specificity of protein-protein interactions are not nearly as standardized as nucleic acid hybridization. The use of lectin microarrays in glycoform analysis also encounters the similar problems. 

TABLE 14.7 Fluorescent dyes used in microarray detection 

Note Structures of some listed fluorescent dyes are  

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Fig. 5. Measurement of the relative amount of protein immobilized in each spot in the array. A p53 protein function microarray probed with a Cy3-labeled anti-His antibody. Quantification of the signal intensity from each spot reveals the relative amounts of recombinant protein present in each spot in the array.

DNA MICROARRAYS PROBING GENE RESPONSES TO 02 LIMITATION IN HYPOXIA-TOLERANT SPECIES... 

Figure 5 Fluorescence image of oligosaccharide microarrays spotted with 10 ohgosaccha-ride probes with identical carbohydrate chips that were separately incubated with each of 3 biotin-labeled lectins (0.1 mg/ml in PBST) for 1 h, washed with PBST 3 times for 5 min each, and stained with 5 pg/ml of Cy3-strptavidin and evaluated by confocal fluorescence microscopy. Fluorescence images of oligosaccharide microarrays probed with (A) concanavah A (Con A), (B) LoTus Tetragonolobus (LT), and (C) Erythrina Gristagalli (EC).

DNA microarrays, or DNA chips consist of thousands of individual DNA sequences arrayed at a high density on a single matrix, usually glass slides or quartz wafers, but sometimes on nylon substrates. Probes with known identity are used to determine complementary binding, thus allowing the analysis of gene expression, DNA sequence variation or protein levels in a highly parallel format. 

Spike-ins are usually RNA transcripts used to calibrate measurements in a DNA microarray experiment. Each spike-in is designed to hybridize with a specific control probe on the target array. Manufacturers of commercially available microarrays typically offer companion RNA spike-ins kits . Known amounts of RNA spike-ins are mixed with the experiment sample during preparation. Subsequently the measured degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements of the sample RNA. 

The ultimate goal of microarray-based expression analysis is to acquire a comprehension of the entire cellular process, in order to exploit and to standardize the multidi-menisional relations between genotype and phenotype. However, an increasingly important parameter, which has not yet been substantially taken into account, is the role of cellular translation. This means that mRNA expression data need to be correlated with the assortment of proteins actually present in the cell. One approach is based on the use of microarrays containing double-stranded DNA probes for the analysis of DNA-protein interaction and, thus, the detection and identification of DNA-binding proteins by means of fluorescence [130] or mass spectrometry analysis [131]. Moreover, substantial efforts are currently under way to develop protein, antibody, or even cell arrays, applicable to the cor-... 

Figure 7.7. Protein microarray on polyvinylidene difluoride filters (PVDF). Proteins are gridded on filter and probed with an antibody to a gene of interest. Protein-ligand interactions could be detected using a labeled ligand as a probe.

Shalon, D., Smith, S. J., and Brown, P. O. (1996). A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Res. 6, 639-645. 

Pennie WD. Use of cDNA microarrays to probe and understand the toxicological consequences of altered gene expression. Toxicol Lett 2000 112-113 473-477. 

Place a microarray slide in a hybridization chamber and pipette 25 Ail of the labeled probe mixture on the slide surface near one end of the microarray print area. 

In contrast to high density arrays low density arrays are made by deposition of pre-synthesized oligonucleotides or proteins on activated surfaces. There are several printing techniques for fabricating microarrays Non-contact biochip arrayers, commonly based on the piezoelectric effect, can apply controlled sub-nanoliter probe volumes to pre-specified locations on the chip surface. Due to the fact that the dispenser does not touch the surface, a non-contact arrayer provides low risk of contamination and is most suitable for printing on soft materials such as hydrogels. 

Flaim CJ, Chien S, Bhatia SN (2005) An extracellular matrix microarray for probing cellular differentiation. Nat Methods 2 119-125... 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

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