A picoliter chamber array for cell-free protein synthesis.
The completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented cross-contamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10,000 on one chip (capacity 150 pl) and 250,000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.
Kinpara
Takeshi
T
School of Materials Science, Japan Advanced Institute of Science and Technology,1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292.
Mizuno
Ryuta
R
Murakami
Yuji
Y
Kobayashi
Masaaki
M
Yamaura
Shouhei
S
Hasan
Quamrul
Q
Morita
Yasutaka
Y
Nakano
Hideo
H
Yamane
Tsuneo
T
Tamiya
Eiichi
E
D002474:Cell-Free System; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000737:chemistry; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D015723:Gene Library; D005898:Glass / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007477:Ions; D008856:Microscopy, Fluorescence; D010100:Oxygen / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods*; D012828:Silicones / Q000737:chemistry; D013997:Time Factors
D004129:Dimethylpolysiloxanes; D007477:Ions; D011108:Polymers; D012828:Silicones; D049452:Green Fluorescent Proteins; C013830:baysilon; D004247:DNA; D010100:Oxygen