HIGHLY SENSITIVE PYROSEQUENCING SYSTEM WITH POLYMER-SUPPORTED ENZYMES FOR ANALYZING MRNA IN A SINGLE CELL
A highly sensitive massively parallel pyrosequencing system employing a gel matrix that immobilizes high-density enzymes in micro-reaction chambers is demonstrated. The system has a metal-coated plastic plate with micro-chambers that contain DNA samples immobilized on beads. Reducing the chamber size to increase the chamber density is important to realize a high-throughput DNA analyzer utilizing a commercial camera. We succeeded in reducing the chamber size to as small as 6. 5 /μm in order to utilize 4 μm beads that support the DNA in the system. However, the use of small beads as DNA supports has a disadvantage in the detection of luminescence because small amounts of DNA produce low levels of pyrophosphates, and therefore low luminescence. We overcame this difficulty by using a gel matrix to increase the enzyme concentration in the reaction chamber. This resulted in luminescence that was one order of magnitude higher than that measured using a conventional method. The techniques here will contribute to achieving a small, inexpensive pyrosequencing system with high throughput.
Pyrosequence gel, photo-reactive polymer enzyme immobilization single-cell analysis
M Shirai T.Kajiyama M.Goto J.Izumisawa Y.Harada H.Kambara
Central Research Lab, Hitachi, Ltd,Tokyo, Japan Central Research Lab, Hitachi, Ltd, Tokyo, Japan R& D Division, Kikkoman Corp, Chiba, Japan
国际会议
The 6th International Forum on Post-genome Technologies(6IFPT)(第六届国际后基因组生命科学技术学术论坛)
北京
英文
340-343
2009-09-17（万方平台首次上网日期，不代表论文的发表时间）