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Novel concept microarray enabling PCR and multistep reactions through pipette-free aperture-to-aperture parallel transfer.

Kinoshita Y, Tayama T, Kitamura K, Salimullah M, Uchida H, Suzuki M, Husimi Y, Nishigaki K - BMC Biotechnol. (2010)

Bottom Line: On the other hand, the popular microplate technology, which has a great merit of being able to perform parallel multistep reactions, has come to its limit in increasing the number of wells (currently, up to 9600) and reducing the volume to deal with due to the difficulty in operations.These were demonstrated by applying the MMV technology to searching lysozyme-crystallizing conditions and selecting peptides aimed for Aβ-binding or cathepsin E-inhibition.With the introduction of a novel concept microarray (MMV) technology, parallel and multistep reactions in sub-μL scale have become possible.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Functional Materials Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Saitama 338-8570, Japan.

ABSTRACT

Background: The microarray has contributed to developing the omic analysis. However, as it depends basically on the surface reaction, it is hard to perform bulk reactions and sequential multistep reactions. On the other hand, the popular microplate technology, which has a great merit of being able to perform parallel multistep reactions, has come to its limit in increasing the number of wells (currently, up to 9600) and reducing the volume to deal with due to the difficulty in operations.

Results: Here, we report a novel microarray technology which enables us to explore advanced applications, termed microarray-with-manageable volumes (MMV). The technical essence is in the pipette-free direct parallel transfer from well to well performed by centrifugation, evading the evaporation and adsorption-losses during handling. By developing the MMV plate, accompanying devices and techniques, generation of multiple conditions (256 kinds) and performance of parallel multistep reactions, including PCR and in vitro translation reactions, have been made possible. These were demonstrated by applying the MMV technology to searching lysozyme-crystallizing conditions and selecting peptides aimed for Aβ-binding or cathepsin E-inhibition.

Conclusions: With the introduction of a novel concept microarray (MMV) technology, parallel and multistep reactions in sub-μL scale have become possible.

Show MeSH
2N method. Each well on an MMV can be addressed using the binary number. For example, in case of 16 wells-MMV (16 = 24, N = 4), address-allotting becomes as shown in the center of this figure. If we regard each order (bit) corresponding to a different element (in this case, color), then 4 different (orthogonal) elements need to be prepared. If we take '1' as 'add' and '0' as 'not add', each binary addressing corresponds to directing which elements should be added there. So, the template way (see Fig. 3c) requires 4 different template plates as shown in this figure. By using these four templates, 16 different states can be prepared in an MMV.
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Figure 5: 2N method. Each well on an MMV can be addressed using the binary number. For example, in case of 16 wells-MMV (16 = 24, N = 4), address-allotting becomes as shown in the center of this figure. If we regard each order (bit) corresponding to a different element (in this case, color), then 4 different (orthogonal) elements need to be prepared. If we take '1' as 'add' and '0' as 'not add', each binary addressing corresponds to directing which elements should be added there. So, the template way (see Fig. 3c) requires 4 different template plates as shown in this figure. By using these four templates, 16 different states can be prepared in an MMV.

Mentions: To generate multiple conditions (256 species), '2N method' was employed using dry MMVs. In case of generating 256 different conditions, 8 species of MMV plates (2N = 256; then N = 8) were used for this purpose (see Fig. 3f and Fig. 5). The transfer was carried out by spinning two face-to-face stacked MMV plates, a recipient MMV (bottom) and a donor MMV (top) (Fig. 3e). Using the MMV thus made, we could obtain four distinctive types of crystals: typical, micro, needle-like and amorphous crystals (Fig. 6) corresponding to the phase diagram in Fig. 7. This result informs us that the conditions of the lower NaCl concentrations (0.2~0.4 M) and the pH of around 4~5 were especially suitable for obtaining a large tetragonal crystalline and that the higher salt concentration (0.9~1.5 M) and the lower pH (pH 3~4) conditions generated the needle-like crystalline whereas the other conditions led to poor generation of crystallines. Such systematic information on crystallization conditions must be very useful for the physicochemical study of morphogenesis mechanism [29-32]. By applying this method, the cost-performance can be greatly improved due to the tiny scale (sub-μL) of this method and, otherwise unavailable experiments must be made possible (see '2N Method' in Methods).


Novel concept microarray enabling PCR and multistep reactions through pipette-free aperture-to-aperture parallel transfer.

Kinoshita Y, Tayama T, Kitamura K, Salimullah M, Uchida H, Suzuki M, Husimi Y, Nishigaki K - BMC Biotechnol. (2010)

2N method. Each well on an MMV can be addressed using the binary number. For example, in case of 16 wells-MMV (16 = 24, N = 4), address-allotting becomes as shown in the center of this figure. If we regard each order (bit) corresponding to a different element (in this case, color), then 4 different (orthogonal) elements need to be prepared. If we take '1' as 'add' and '0' as 'not add', each binary addressing corresponds to directing which elements should be added there. So, the template way (see Fig. 3c) requires 4 different template plates as shown in this figure. By using these four templates, 16 different states can be prepared in an MMV.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959086&req=5

Figure 5: 2N method. Each well on an MMV can be addressed using the binary number. For example, in case of 16 wells-MMV (16 = 24, N = 4), address-allotting becomes as shown in the center of this figure. If we regard each order (bit) corresponding to a different element (in this case, color), then 4 different (orthogonal) elements need to be prepared. If we take '1' as 'add' and '0' as 'not add', each binary addressing corresponds to directing which elements should be added there. So, the template way (see Fig. 3c) requires 4 different template plates as shown in this figure. By using these four templates, 16 different states can be prepared in an MMV.
Mentions: To generate multiple conditions (256 species), '2N method' was employed using dry MMVs. In case of generating 256 different conditions, 8 species of MMV plates (2N = 256; then N = 8) were used for this purpose (see Fig. 3f and Fig. 5). The transfer was carried out by spinning two face-to-face stacked MMV plates, a recipient MMV (bottom) and a donor MMV (top) (Fig. 3e). Using the MMV thus made, we could obtain four distinctive types of crystals: typical, micro, needle-like and amorphous crystals (Fig. 6) corresponding to the phase diagram in Fig. 7. This result informs us that the conditions of the lower NaCl concentrations (0.2~0.4 M) and the pH of around 4~5 were especially suitable for obtaining a large tetragonal crystalline and that the higher salt concentration (0.9~1.5 M) and the lower pH (pH 3~4) conditions generated the needle-like crystalline whereas the other conditions led to poor generation of crystallines. Such systematic information on crystallization conditions must be very useful for the physicochemical study of morphogenesis mechanism [29-32]. By applying this method, the cost-performance can be greatly improved due to the tiny scale (sub-μL) of this method and, otherwise unavailable experiments must be made possible (see '2N Method' in Methods).

Bottom Line: On the other hand, the popular microplate technology, which has a great merit of being able to perform parallel multistep reactions, has come to its limit in increasing the number of wells (currently, up to 9600) and reducing the volume to deal with due to the difficulty in operations.These were demonstrated by applying the MMV technology to searching lysozyme-crystallizing conditions and selecting peptides aimed for Aβ-binding or cathepsin E-inhibition.With the introduction of a novel concept microarray (MMV) technology, parallel and multistep reactions in sub-μL scale have become possible.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Functional Materials Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Saitama 338-8570, Japan.

ABSTRACT

Background: The microarray has contributed to developing the omic analysis. However, as it depends basically on the surface reaction, it is hard to perform bulk reactions and sequential multistep reactions. On the other hand, the popular microplate technology, which has a great merit of being able to perform parallel multistep reactions, has come to its limit in increasing the number of wells (currently, up to 9600) and reducing the volume to deal with due to the difficulty in operations.

Results: Here, we report a novel microarray technology which enables us to explore advanced applications, termed microarray-with-manageable volumes (MMV). The technical essence is in the pipette-free direct parallel transfer from well to well performed by centrifugation, evading the evaporation and adsorption-losses during handling. By developing the MMV plate, accompanying devices and techniques, generation of multiple conditions (256 kinds) and performance of parallel multistep reactions, including PCR and in vitro translation reactions, have been made possible. These were demonstrated by applying the MMV technology to searching lysozyme-crystallizing conditions and selecting peptides aimed for Aβ-binding or cathepsin E-inhibition.

Conclusions: With the introduction of a novel concept microarray (MMV) technology, parallel and multistep reactions in sub-μL scale have become possible.

Show MeSH