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Highly sensitive pyrosequencing system with polymer-supported enzymes for high-throughput DNA analysis.
Anal Chem. 2011 Aug 21;
Authors: Shiari M, Goto M, Suzuki S, Kono K, Kajiyama T, Kambara H
Abstract
A highly sensitive massively parallel pyrosequencing system employing a gel matrix to immobilize enzymes at high density in micro-reaction chambers is demonstrated. Reducing the size of micro-reaction chambers in a DNA analyzer is important to achieve a high-throughput utilizing a commercially available detection device or camera. A high-performance system can be attained by detecting signals from one reaction chamber with one photo-pixel of around several micrometers by utilizing 1:1 image magnification. However, the use of small beads immobilizing DNA has a disadvantage in detecting luminescence because only small amounts of DNA can be immobilized on the bead surfaces for sequencing. As luminescence intensity could be enhanced by increasing luciferase density in chambers, we overcame this difficulty by using a gel matrix to immobilize luciferase at a high concentration in micro-reaction chambers. One order of magnitude higher luminescence could be observed with the new method compared to the conventional method. Consequently, the chamber size and bead size immobilizing DNA could be reduced to as small as 6.5 ?m and 4-?m, respectively. This can be successfully applied to achieving small, inexpensive pyrosequencing systems with high throughput.
PMID: 21854050 [PubMed - as supplied by publisher]
More...
Highly sensitive pyrosequencing system with polymer-supported enzymes for high-throughput DNA analysis.
Anal Chem. 2011 Aug 21;
Authors: Shiari M, Goto M, Suzuki S, Kono K, Kajiyama T, Kambara H
Abstract
A highly sensitive massively parallel pyrosequencing system employing a gel matrix to immobilize enzymes at high density in micro-reaction chambers is demonstrated. Reducing the size of micro-reaction chambers in a DNA analyzer is important to achieve a high-throughput utilizing a commercially available detection device or camera. A high-performance system can be attained by detecting signals from one reaction chamber with one photo-pixel of around several micrometers by utilizing 1:1 image magnification. However, the use of small beads immobilizing DNA has a disadvantage in detecting luminescence because only small amounts of DNA can be immobilized on the bead surfaces for sequencing. As luminescence intensity could be enhanced by increasing luciferase density in chambers, we overcame this difficulty by using a gel matrix to immobilize luciferase at a high concentration in micro-reaction chambers. One order of magnitude higher luminescence could be observed with the new method compared to the conventional method. Consequently, the chamber size and bead size immobilizing DNA could be reduced to as small as 6.5 ?m and 4-?m, respectively. This can be successfully applied to achieving small, inexpensive pyrosequencing systems with high throughput.
PMID: 21854050 [PubMed - as supplied by publisher]
More...