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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
Advantages of the MMV (microarray-with-manageable volumes). In the conventional microarray, reaction products diffuse away (Panel a) while in the MMV each reaction product can stay in the same site (well) (Panel b). Another merit of the MMV is that each well of an MMV can be treated individually, leading to parallel multistep reactions. In these experiments, the volume of an MMV well can be changed quantitatively as shown in this figure.
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Figure 1: Advantages of the MMV (microarray-with-manageable volumes). In the conventional microarray, reaction products diffuse away (Panel a) while in the MMV each reaction product can stay in the same site (well) (Panel b). Another merit of the MMV is that each well of an MMV can be treated individually, leading to parallel multistep reactions. In these experiments, the volume of an MMV well can be changed quantitatively as shown in this figure.

Mentions: In this stream, we have devised a multi-well type microarray (open system) made of plastics (dry) and gel (wet) and developed its operational method through pilot experiments such as an arrayed-colony formation, a parallel monitoring of various conditions (for crystallization), and an application to multistep reactions. To develop such technologies, fabricating microarrays in-house was actually prerequisite. As is well-known, the conventional microarray technology has been developed based on the highly sophisticated photo-lithographic technology developed for the IC industry [20]. Therefore, the technological framework was well-established and rather easy to attain a high density of arrays (e.g., millions per square inches). Though the IC-derived technology is good at surface reactions, this technology developed for handling 'information (state)' but not 'materials (molecules)' has a limitation in being applied to a bulk reaction of molecules. One typical inconvenience is, as shown in Fig. 1, in the inability to succeed individual reaction products at each spot for the next reaction independently, thus making the parallel multistep reactions impossible. Since almost all reactions in vitro need to be multistep ones, a number of separate tubes (or their collective form termed as microplate) have been traditionally used with an aid of robotics, resulting in a massive system [21-23]. Currently, the number of wells per micro-plate has boosted up to almost the limit of 3456 to 9600 with increase of difficulties in operation time and throughput accuracy [24]. In this paper, a novel method depending on a wet and dry microarray system was developed, which enables parallel operations of sub-μL aliquots by overcoming difficult problems such as measuring and transferring sub-μL solutions in parallel without critical evaporation and adsorption-losses of samples. As a result, multi-different conditions were formed for crystallization of proteins and multistep reactions were successively performed in parallel beginning with PCR followed by transcription, translation, modifying enzyme reaction, and protease-activity assay. Thus, this paper reports a great potentiality of a novel concept microarray MMV for the first time.


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)

Advantages of the MMV (microarray-with-manageable volumes). In the conventional microarray, reaction products diffuse away (Panel a) while in the MMV each reaction product can stay in the same site (well) (Panel b). Another merit of the MMV is that each well of an MMV can be treated individually, leading to parallel multistep reactions. In these experiments, the volume of an MMV well can be changed quantitatively as shown in this figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Advantages of the MMV (microarray-with-manageable volumes). In the conventional microarray, reaction products diffuse away (Panel a) while in the MMV each reaction product can stay in the same site (well) (Panel b). Another merit of the MMV is that each well of an MMV can be treated individually, leading to parallel multistep reactions. In these experiments, the volume of an MMV well can be changed quantitatively as shown in this figure.
Mentions: In this stream, we have devised a multi-well type microarray (open system) made of plastics (dry) and gel (wet) and developed its operational method through pilot experiments such as an arrayed-colony formation, a parallel monitoring of various conditions (for crystallization), and an application to multistep reactions. To develop such technologies, fabricating microarrays in-house was actually prerequisite. As is well-known, the conventional microarray technology has been developed based on the highly sophisticated photo-lithographic technology developed for the IC industry [20]. Therefore, the technological framework was well-established and rather easy to attain a high density of arrays (e.g., millions per square inches). Though the IC-derived technology is good at surface reactions, this technology developed for handling 'information (state)' but not 'materials (molecules)' has a limitation in being applied to a bulk reaction of molecules. One typical inconvenience is, as shown in Fig. 1, in the inability to succeed individual reaction products at each spot for the next reaction independently, thus making the parallel multistep reactions impossible. Since almost all reactions in vitro need to be multistep ones, a number of separate tubes (or their collective form termed as microplate) have been traditionally used with an aid of robotics, resulting in a massive system [21-23]. Currently, the number of wells per micro-plate has boosted up to almost the limit of 3456 to 9600 with increase of difficulties in operation time and throughput accuracy [24]. In this paper, a novel method depending on a wet and dry microarray system was developed, which enables parallel operations of sub-μL aliquots by overcoming difficult problems such as measuring and transferring sub-μL solutions in parallel without critical evaporation and adsorption-losses of samples. As a result, multi-different conditions were formed for crystallization of proteins and multistep reactions were successively performed in parallel beginning with PCR followed by transcription, translation, modifying enzyme reaction, and protease-activity assay. Thus, this paper reports a great potentiality of a novel concept microarray MMV for the first time.

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