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 mu m in order to utilize 4 mu m 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.