1. Hybridization has several different meanings in science:

• In biological sciences, it is the process of mixing different species or varieties of organisms to create a hybrid or the joining two complementary strands of DNA.
• In chemical sciences, orbital hybridization is the mixing of atomic orbitals to form new orbitals suitable for bonding.

Creating a hybrid strand of DNA or RNA
Two strands of a DNA molecule are denatured by heating to about 100°C. At this temperature, the complementary base pairs that hold the double helix strands together are disrupted and the helix rapidly dissociates into two single strands. The DNA denaturation is reversible by keeping the two single stands of DNA for a prolonged period at 65°C = 149°F. This process is called DNA renaturation or hybridization. Similar hybridization reactions can occur between any single stranded nucleic acid chain: DNA/DNA, RNA/RNA, DNA/RNA. If an RNA transcript is introduced during the hybridization process, the RNA competes with the coding DNA strand and forms double-stranded DNA/RNA hybrid molecules. These hybridization reactions can be used to detect and characterize nucleotide sequences using a particular nucleotide sequence as a probe.

The term ‘genomics’ refers to the comprehensive study of genes and their function. Recent advances in bioinformatics and high-throughput technologies, such as microarray analysis, are bringing about a revolution in our understanding of the molecular mechanisms underlying normal and dysfunctional biological processes. Microarray studies and other genomic techniques are also stimulating the discovery of new targets for the treatment of disease, which is aiding drug development, immunotherapeutics and gene therapy.

2. DNA microarrays

a.) Gene expression profiling or microarray analysis has enabled the measurement of thousands of genes in a single RNA sample.

There are a variety of microarray platforms that have been developed to accomplish this and the basic idea for each is simple: a glass slide or membrane is spotted or "arrayed" with DNA fragments or oligonucleotides that represent specific gene coding regions. Purified RNA is then fluorescently or radioactively labelled and hybridized to the slide/membrane (figure 1). In some cases, hybridization is done simultaneously with reference RNA to facilitate comparison of data across multiple experiments. After thorough washing, the raw data is obtained by laser scanning or autoradiographic imaging (figure 1). At this point, the data may be entered into a database and analyzed by a number of statistical methods.

Figure 1: Principle of DNA-microarrays

Conventional microscope slides (75 mm by 25 mm) are one of the most common microarray substrates used. Up to several thousand spots of oligonucleotides or cDNA probes with known identity cover the slide in a checkerboard pattern. In the standard experimental set-up the sample is sandwiched between the DNA microarray and a cover slip forming a reaction chamber with an area of several cm² and a height of only 20–100 µm. Using this setup, diffusion is the only mechanism by which DNA strands move within the capillary gap.

However, diffusion is a notoriously slow process for molecules the size of DNA strands, which may need to travel a distance of several centimetres to their complementary spots. Using this set-up can lead to rapid depletion of rare or low copy number cDNA molecules since the spots closest to the entry point will bind them quickly, leaving the last spots to be reached with little or no chance of binding any. This diffusion limitation leads to unbalanced results.

As a result, diffusion limitation leads to low signal-to-noise ratios as only a fraction of the molecules present in the sample have a chance to bind to their complimentary spots. Also, the slide-to-slide reproducibility, as well as the homogeneity, of replicate spots across the slide are significantly reduced. Furthermore, microarray experiments are generally left to hybridize overnight and yet this is not enough time for a diffusion-based system to reach homogenous distribution, and hence microarray reproducibility is often low. In order to guarantee fast and reproducible hybridization results the diffusion limit must be overcome by agitation.

The SlideBooster with its SAW (surface acoustic wave) micro-agitation chips efficiently agitates even the smallest sample volumes (even below 10 µL) without introducing any dead volume. As a result there is a reduced reaction time, increased signal-to-noise-ratio and improved homogeneity across the microarray.

Please refer to:
1. Publication: Enhancing results of microarray hybridizations through microagitation (2003)
2. Application Report: Genisphere Kit, Download pdf (308 kb)
3. Figure 2

Figure 2: Pseudocolour images of microarrays incubated with different protocols. Each array contains four replicates, two adjacent in each of the two subarrays.

3. Comparative genomic hybridization

Comparative genomic hybridization (CGH) is a molecular cytogenetic method for the detection of chromosomal imbalances. In a CGH experiment, two genomic DNA samples are simultaneously hybridized in situ to normal human metaphase spreads, and detected with different fluorochromes. The intensity ratio of the two fluorescence signals gives a measure for the copy number ratio between the two genomic DNA samples. CGH can be either performed on metaphase chromosomes or using BAC DNA as array-CGH (figure 3)

Array–CGH: A grid of well-defined, genomically mapped DNA fragments (e.g. BAC clones, cDNA, oligonucleotides) immobilized on a glass surface replace condensed metaphase chromosomes as the hybridization target.

As a result, the resolution achieved can be dramatically increased, since it depends on the number of gene fragments represented and their separation distance. Currently, whole genome arrays with resolutions from 1 Mb to 150 kb and even in the range of single genes or exons are available. These high resolutions permit the detection of sub-microscopic genomic imbalances, as well as the precise breakpoint determination for genomic aberrations. Therefore, array-CGH can complement the standard cytogenetic methods (chromosome analysis, subtelomere analysis, FISH) in prenatal, postnatal and tumour-cytogenetic analysis. In addition, array-CGH enables the sequencebased mapping of aberrations via the identification of chromosomal segments, showing copy number aberrations (deletion, duplication or amplification).

Figure 3: Comparison of conventional CGH and array-CGH
Origin: www.array-cgh.de

The main requirements for cytogenetic applications are standardized and reproducible experimental conditions, with the highest possible sensitivity. Mixing during hybridization and automated washing to a sensible degree, guarantee standardized user-independent experimental conditions with minimal variation, whilst offering an uncomplicated setup, ease of use and maintenance-free operation. In addition, the assay time is considerably reduced (down to 24 hours) by the mixing, ensuring the implementation of array-CGH into the daily laboratory workflow, more feasible and calculable. The SlideBooster in combination with the AdvaWash offers the necessary features to achieve the highest possible sensitivity with a shortened incubation time.

Please refer to:
1. Application Report: Array-CGH, Download pdf (299 kb)
2. Figure 4

Figure 4: Scan of a CGH-slide incubated with the SlideBooster for 24 h (42°C, 27dbm, pulse/pause 5:5)
Green Signal: microdeletion in patient’s DNA. Hybridization only by the green labelled reference DNA.
Red Signal: microamplification in patient’s DNA. Hybridization only by the red labeled patient’s DNA.
Yellow Signal: no changes in patient’s DNA. Red and green labeled DNA is hybridizing.
Origin: http://www.array-cgh.de/

4. Protein microarrays

A protein microarray consists of a substrate (typically made from glass) on which different protein molecules are placed at separate locations in an ordered manner forming a microscopic array. They are used to identify protein-protein interactions, such as the substrates of protein kinases, or the targets of biologically active small molecules.

Figure 5: Samples of antibody microarray creations and detections
Origin: http://en.wikipedia.org/wiki/Antibody_microarray

The most common protein microarray format uses antibodies, where antibodies are spotted onto the protein chip and are used as capture molecules to detect proteins from cell lysates. Figure 5 shows several methods of creating and detecting antibody microarrays.

Assay systems that employ protein microarrays for the analysis of complex samples are powerful tools that generate large quantities of data from a limited amount of sample. One downside of the miniaturization of these systems though, is that they are susceptible to fluctuations during signal generation. Also, the use of uniform conditions during sample incubation and assay procedure is required in order to get reproducible results.

As discussed above, diffusion limits prevent homogenous distribution of the molecules and lead to a decrease in array sensitivity. Using the incubation stations with the Beckman Coulter microagitation technology (SAW), enables increased signal, shortened incubation time or a decrease in the required sample concentration.

Please refer to:
1. Publications:

• Increasing robustness and sensitivity of protein microarrays through microagitation and automation (2006)
• Kinetics of antigen binding to antibody microspots: Strong limitations by mass transport to the surface (2006)

2. Figure 6

Figure 6: Human IgG assay: 2 positive (rows a, b) and 3 negative (rows c, d, e) controls with 4 replicas each. The three-dimensional bar graph shows the mean intensities of row b for various experimental parameters.

5. Immunohistochemistry

Immunohistochemistry (IHC) uses the principle of antibody/antigen binding to identify and localize proteins of interest within the cells of a tissue section. To enable this, the antibodies are directly or indirectly labelled with a chromophore or fluorophore. IHC is a key technique in research as well as investigative and diagnostic pathology, where there is a need to correlate the localization of antigens in specific cells with tissue morphology.

There are two strategies used for the immmunohistochemical detection of antigens in tissue, the direct and the indirect labelling methods.

The direct method (figure 7) uses one labelled antibody, which binds directly to the antigen being targeted. This technique utilizes only one antibody and the procedure is therefore simple and rapid. However, it can suffer problems with sensitivity due to the limited signal amplification and is subsequently not used as much as the indirect method. The primary antibody can be labelled with a fluorescent dye or an enzyme.

Figure 7: The direct method of immunohistochemical labelling uses one labelled antibody, which binds directly to the antigen being targeted. Origin: http://en.wikipedia.org/wiki/Immunohistochemistry *


The indirect method (figure 8) involves an unlabelled primary antibody (first layer) which reacts with the tissue antigen, and a labelled secondary antibody (second layer) which reacts with the primary antibody. (The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised, e.g. an antibody raised in a goat against rabbit IgG) This method is more sensitive since there is a signal amplification process as a number of secondary antibodies can bind to each primary antibody at different antigenic sites. The secondary antibody can be labelled with a fluorescent dye or an enzyme.

Figure 8: The indirect method of immunohistochemical labelling uses one antibody against the antigen being probed for and a second, labelled, antibody against the first. Origin http://en.wikipedia.org/wiki/Immunohistochemistry *

* Diagram, created 14th September 2005, by Viki MaleI, the creator of this work, hereby grant the permission to copy, distribute and/or modify this document under the terms of the GNU Free Documentation License, Version 1.2 or any later version published by the Free Software Foundation; with no Invariant Sections, no Front-Cover Texts, and no Back-Cover Texts Subject to disclaimers.

Sensitivity is crucial during IHC or ISH (in situ hybridization) and this is limited by the diffusion and the amount of labelled antibody. Diffusion is the only mechanism by which the antibodies and substrates are transported to their targets. Thus by overcoming the diffusion limitation with microagitation (such as by using the Beckman Coulter incubation stations) the sensitivity will be significantly increased. The benefit of increasing the sensitivity of IHC is that the amount of antibody and subsequent cost can be reduced. This means, for example that higher dilutions can be used to yield better results, since there will be less background staining – this is especially important for many polyclonal antibodies.
Several comparisons between methods using the SlideBooster and standard manual methods with antibody incubation in hybridisation ovens, show that antibody concentrations can be reduced five-fold without any loss in sensitivity, destruction of the tissue sections and can also enable shortened incubation times

Please refer to:
1. Application Report: IHC staining, Download pdf (234 kb)
2. Publication: High sensitivity and reproducibility of immunohistochemistry with microagitation (2004)
3. Figure 9 and 10

Figure 9: IHC staining with SAW-agitation (left) and without agitation (right)

Figure 10: IHC staining with SAW-agitation (left) and without agitation (right)

6. Fluorescence in situ hybridization

Fluorescence in situ hybridzation (FISH) is a cytogenetic technique which can be used to detect and localise the presence or absence of specific DNA sequences on chromosomes. It uses fluorescent probes which bind only to those parts of the chromosome with which they show a high level of sequence similarity. Fluorescence microscopy is then commonly used to detail where the fluorescent probes have bound to the chromosomes.

Figure 11: Model of fluorescence in situ hybridization
Origin: http://en.wikipedia.org/wiki/Fluorescent_in_situ_hybridization

FISH can be used to

• map sequences to a specific position on a chromosome.
• do chromosome painting to make a comparison between two species or varieties by using DNA from entire chromosomes or even the entire genome of one species/variety as a probe on the other. In this way, Chromosomal abnormalities can be identified and evolutionary relations can be deduced.
• identify micro-organisms, which is widely used in the field of microbial ecology. Biofilms, for example, are complex (often) multi-species bacterial organisations. Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the distribution of this specific species in the biofilm. Preparing probes (in two different colours) for two species allows the visualization/study of the co-localization of these two species in the biofilm, revealing the fine architecture of the biofilm.

Saving assay time and reducing reagent costs are the key requirements of all new approaches focusing on improvement of these methods. As diffusion is the only mechanism presently used to ensure that probes reach their targets, microagitation can be used to greatly improve the results in terms of reproducibility and sensitivity.
Several comparisons between the SlideBooster and a standard manual method using hybridization ovens show that a dramatic reduction in incubation time is possible with the SlideBooster without any reduction in quality.

Please refer to:
1. Table 1
2. Figure 12

 
Table 1: Results of different comparisons SlideBooster vs. conventional method



Figure 12: Result of chromosomal FISH incubated in SlideBooster for 2 h

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