Hybridization-based approaches

The transcriptome is defined as the whole set of transcripts present in a cell in a given developmental stage and under a specific physiological condition. As we have been describing, the transcriptome is composed by mRNAs, rRNAs, tRNAs, and a set of ncRNAs (22,102). Over the past decade, the transcriptome has been analyzed by hybridization-based approaches (microarrays). Typically, the RNA is previously converted into cDNA, fluorescently labeled, and hybridized onto a specific microarray (such as Affymetrix GeneChips). They have been widely used over the past 15 years and have given quite precise information about the way the transcriptome is regulated... 

OLA. The OLA uses an enzymatic reaction to increase the specificity of a hybridization-based approach. Three very specific oligonucleotide probes are used in OLA one specific for the wild-type allele, one specific for the variant allele, and a common probe that carries a fluorescent label. PCR is used to create amplicons containing the polymorphic site. When the PCR products are incubated with all three probes, the 5 region of the common probe anneals just downstream of the polymorphic site. The 3 end of either of the allele specific probes anneals adjacent to the 5 end of the common probe. In the presence of thermostable DNA ligase, the two probes will join only if there is a perfect match. The results of the assay can be observed either by gel... 

A hybridization-based approach has also been developed to quantitate levels of ribozymes in serum using paired complementary oligonucleotide probes, one labeled with biotin and the other with digoxigenin [90]. The annealed triplex is first collected on streptavidin-coated 96-well plates, then an anti-digoxigenin antibody conjugated with alkaline phosphatase is added, followed by addition of the enzyme substrate p-nitrophenyl phosphate, which is cleaved into a soluble colored product that is quantitated by absorbance at 405 nm. 

Toby G, Golemis E. Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions. Methods 2001 24 201-217. 

Literature in the area of neural networks has been expanding at an enormous rate with the development of new and efficient algorithms. Neural networks have been shown to have enormous processing capability and the authors have implemented many hybrid approaches based on this technique. The authors have implemented an ANN based approach in several areas of polymer science, and the overall results obtained have been very encouraging. Case studies and the algorithms presented in this chapter were very simple to implement. With the current expansion rate of new approaches in neural networks, the readers may find other paradigms that may provide new opportunities in their area of interest. 

Other approximate, more empirical methods are the extended Huckel 31> and hybrid-based Hiickel 32. 3> approaches. In these methods the electron repulsion is not taken into account explicitly. These are extensions of the early Huckel molecular orbitals 4> which have successfully been used in the n electron system of planar molecules. On account of the simplest feature of calculation, the Hiickel method has made possible the first quantum mechanical interpretation of the classical electronic theory of organic chemistry and has given a reasonable explanation for the chemical reactivity of sizable conjugated molecules. 

Although solution-based approaches can be used as a direct replacement for vacuum-based processes, one of the key aspects of atmospheric solution-based processing is that, by virtue of it being non vacuum and using conformal solution-based precursors, it can lead to a next generation of hybrid and nanostructured materials and devices by enabling unique composites... 

The case study from chapter 7 is concerned with the design and improvement of chemically-active ship bottom paints known as antifouling paints. A hybrid experiment-model based approach is employed here. Experiments and use of expert knowledge are employed to identify product alternatives, whose evaluation in terms of performance as a marine biofouling protector is verified through a model-based approach. 

In the field of process synthesis, there are basically three different approaches available (1) mathematical optimization based approaches, (2) heuristic based approaches, and (3) hybrid approaches... 

The hybrid approach tries to combine the model-based optimization approach with the heuristic approach, thereby, avoiding the problems of unavailability of models. In the initial stages, a property-based approach (where properties are obtained through model or experimental measurements) is applied and in the final stages (where models are usually easy to develop), a model-based optimization approach is applied (see for example, Gani (2004)). More work is needed to establish this technique for food-process applications. 

Basically, two types of approaches are developed here iterative (optimization-based) approaches like the one by Sippl et al. [101] and direct approaches like the one by Kabsch [102, 103], based on Lagrange multipliers. Unfortunately, the much expedient direct methods may fail to produce a sufficiently accurate solution on some degenerate cases. Redington [104] suggested a hybrid method with an improved version of the iterative approach, which requires the computation of only two 3x3 matrix multiplications in the inner loop of the optimization. 

Steps 2 and 5 require relatively little computer time. As for the transition-based approach, step 3 can require significant computational effort if a hybrid functional is used and otherwise requires negligible effort. Step 4 is the most expensive step and will need to be repeated for enough values of u> to sufficiently cover the range of energies of interest. 

Alternative microarray approaches are available that involve the 3 tailing of the miRNA molecule with poly(A), followed by labeling and hybridization. The NCode miRNA microarray platform (Life Technologies) involves the addition of the poly(A) tail to the miRNA prior to ligation of fluorescent dye molecules and subsequent hybridization to the antisense miRNA probes on the microarray chip. In the case of Affymetrix GeneChip microRNA arrays, the poly(A)-tailed miRNAs are labeled with a biotinylated signal molecule. A limitation of hybridization-based microarrays is that because of the small size of miRNAs, the Tm of the probes has a wide range that may diminish specificity and/or sensitivity for miRNAs with a low GC content (10,56). 

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