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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
Selection of Aβ-binding peptides performed in a 1000-wells MMV. (a) Those wells which contain Aβ-binding peptides are indicated by an arrow (bright wells due to the fluorescence of the fused GFP). (b) DNA construct of the variable peptide-library. PT7 designates T7 promoter and franked by other regulatory sequences for transcription/translation of the gene. GFP means the green fluorescent protein gene. GFP is used for detection of peptide binding to Aβ on the magnetic beads. His tag and FLAG region are used for purification and detection of the protein. Xa is the recognition sequence for the restriction cutting of protease Xa. Variable region encodes the sequence of various peptides.
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Figure 9: Selection of Aβ-binding peptides performed in a 1000-wells MMV. (a) Those wells which contain Aβ-binding peptides are indicated by an arrow (bright wells due to the fluorescence of the fused GFP). (b) DNA construct of the variable peptide-library. PT7 designates T7 promoter and franked by other regulatory sequences for transcription/translation of the gene. GFP means the green fluorescent protein gene. GFP is used for detection of peptide binding to Aβ on the magnetic beads. His tag and FLAG region are used for purification and detection of the protein. Xa is the recognition sequence for the restriction cutting of protease Xa. Variable region encodes the sequence of various peptides.

Mentions: First, the selection of Aβ-binding peptides was performed, consisting of 5 consecutive steps of reaction: PCR-amplification of DNA, in vitro transcription, in vitro translation, binding of Aβ and Aβ-binding peptides, and monitoring the fluorescence of GFP (which is expressed as a protein fused with Aβ-binding peptides, thus serving as a marker) with three exchanges of MMV plates. The final result of these reactions is the Aβ-binding peptides as shown in Fig. 9 where those wells which contain GFP-fused peptides trapped by Aβ on a magnetic bead are illuminating (dark wells). Those wells where GFPs are expressed and halted (Aβ-binding peptides with a sufficient binding affinity with Aβ can be expected there) indicate the success of a series of reactions. The background brightness is caused by the reflection of the excitation beam on the surface of sample solutions. In this experiment, the peptide sequences could be obtained from the corresponding DNAs contained in the same well by PCR (data obtained are shown in Table 1).


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)

Selection of Aβ-binding peptides performed in a 1000-wells MMV. (a) Those wells which contain Aβ-binding peptides are indicated by an arrow (bright wells due to the fluorescence of the fused GFP). (b) DNA construct of the variable peptide-library. PT7 designates T7 promoter and franked by other regulatory sequences for transcription/translation of the gene. GFP means the green fluorescent protein gene. GFP is used for detection of peptide binding to Aβ on the magnetic beads. His tag and FLAG region are used for purification and detection of the protein. Xa is the recognition sequence for the restriction cutting of protease Xa. Variable region encodes the sequence of various peptides.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Selection of Aβ-binding peptides performed in a 1000-wells MMV. (a) Those wells which contain Aβ-binding peptides are indicated by an arrow (bright wells due to the fluorescence of the fused GFP). (b) DNA construct of the variable peptide-library. PT7 designates T7 promoter and franked by other regulatory sequences for transcription/translation of the gene. GFP means the green fluorescent protein gene. GFP is used for detection of peptide binding to Aβ on the magnetic beads. His tag and FLAG region are used for purification and detection of the protein. Xa is the recognition sequence for the restriction cutting of protease Xa. Variable region encodes the sequence of various peptides.
Mentions: First, the selection of Aβ-binding peptides was performed, consisting of 5 consecutive steps of reaction: PCR-amplification of DNA, in vitro transcription, in vitro translation, binding of Aβ and Aβ-binding peptides, and monitoring the fluorescence of GFP (which is expressed as a protein fused with Aβ-binding peptides, thus serving as a marker) with three exchanges of MMV plates. The final result of these reactions is the Aβ-binding peptides as shown in Fig. 9 where those wells which contain GFP-fused peptides trapped by Aβ on a magnetic bead are illuminating (dark wells). Those wells where GFPs are expressed and halted (Aβ-binding peptides with a sufficient binding affinity with Aβ can be expected there) indicate the success of a series of reactions. The background brightness is caused by the reflection of the excitation beam on the surface of sample solutions. In this experiment, the peptide sequences could be obtained from the corresponding DNAs contained in the same well by PCR (data obtained are shown in Table 1).

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