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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
How to generate and operate MMVs. (a) MMV generator. (b) A projection pattern used for 1000 well type (actually 988 wells for samples and 3 guide holes). (c) An example of a plastic MMV (1024 well type). (d) Procedures for generating an MMV. Namely, i) place a bedding plate (plastics or pre-molded gel) in a container, ii) pour the monomer solution (for either plastics or gel formation), iii) irradiate the pattern-carrying light (see Fig. 2b), iv) remove the non-polymerized solution (blank) and, finally, v) get an MMV (yellow).
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Figure 2: How to generate and operate MMVs. (a) MMV generator. (b) A projection pattern used for 1000 well type (actually 988 wells for samples and 3 guide holes). (c) An example of a plastic MMV (1024 well type). (d) Procedures for generating an MMV. Namely, i) place a bedding plate (plastics or pre-molded gel) in a container, ii) pour the monomer solution (for either plastics or gel formation), iii) irradiate the pattern-carrying light (see Fig. 2b), iv) remove the non-polymerized solution (blank) and, finally, v) get an MMV (yellow).

Mentions: Following the methods described in Methods, the MMV was prepared and operated (see Fig. 2). MMVs were fabricated using the apparatus built in-house which is composed of DMD (Digital Multi-mirror Device) and others (Fig. 2a) [25,26]. We could form any type of vessels, filters, templates, and others made of either plastics or gel through light-induced polymerization by controlling the light pattern generated by DMD (Fig. 2b-d). MMVs made of gel (wet type) were first introduced here and used in combination with plastics MMVs (dry type) to fulfill multistep reactions as described in Methods. The basic operation of sample transfer from one to another MMV is an aperture-to-aperture way as shown in Fig. 3a and Fig. 3e. In order to demonstrate the feasibility of multistep reactions in the MMV, DNA encoding green fluorescent protein (GFP) was PCR-amplified using an MMV (see 'MMV operations' in Methods and Additional file 1). The amplified DNA molecules were transferred to another MMV, partly filled with a solution for the transcription/translation reaction, and then subjected to the reaction. The resulting checker pattern of fluorescent GFP proteins (see Fig. 4) confirmed the success of the parallel aperture-to-aperture transfer and the series of reactions: PCR, transcription, and translation which finally generate fluorescent GFPs (see 'Verification of the MMV transfer operation' 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)

How to generate and operate MMVs. (a) MMV generator. (b) A projection pattern used for 1000 well type (actually 988 wells for samples and 3 guide holes). (c) An example of a plastic MMV (1024 well type). (d) Procedures for generating an MMV. Namely, i) place a bedding plate (plastics or pre-molded gel) in a container, ii) pour the monomer solution (for either plastics or gel formation), iii) irradiate the pattern-carrying light (see Fig. 2b), iv) remove the non-polymerized solution (blank) and, finally, v) get an MMV (yellow).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: How to generate and operate MMVs. (a) MMV generator. (b) A projection pattern used for 1000 well type (actually 988 wells for samples and 3 guide holes). (c) An example of a plastic MMV (1024 well type). (d) Procedures for generating an MMV. Namely, i) place a bedding plate (plastics or pre-molded gel) in a container, ii) pour the monomer solution (for either plastics or gel formation), iii) irradiate the pattern-carrying light (see Fig. 2b), iv) remove the non-polymerized solution (blank) and, finally, v) get an MMV (yellow).
Mentions: Following the methods described in Methods, the MMV was prepared and operated (see Fig. 2). MMVs were fabricated using the apparatus built in-house which is composed of DMD (Digital Multi-mirror Device) and others (Fig. 2a) [25,26]. We could form any type of vessels, filters, templates, and others made of either plastics or gel through light-induced polymerization by controlling the light pattern generated by DMD (Fig. 2b-d). MMVs made of gel (wet type) were first introduced here and used in combination with plastics MMVs (dry type) to fulfill multistep reactions as described in Methods. The basic operation of sample transfer from one to another MMV is an aperture-to-aperture way as shown in Fig. 3a and Fig. 3e. In order to demonstrate the feasibility of multistep reactions in the MMV, DNA encoding green fluorescent protein (GFP) was PCR-amplified using an MMV (see 'MMV operations' in Methods and Additional file 1). The amplified DNA molecules were transferred to another MMV, partly filled with a solution for the transcription/translation reaction, and then subjected to the reaction. The resulting checker pattern of fluorescent GFP proteins (see Fig. 4) confirmed the success of the parallel aperture-to-aperture transfer and the series of reactions: PCR, transcription, and translation which finally generate fluorescent GFPs (see 'Verification of the MMV transfer operation' 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