Experimental Principles (Sequencing/454 GS FLX)

We carry out sequencing with the 454 GS FLX. A library is prepared from randomly fragmented DNA of a predetermined length. After clonal amplification of the library DNA on each bead by emulsion PCR, base sequences are determined by means of pyrosequencing.
Sample quality check

PicoGreen is used to measure the concentration, and electrophoresis to confirm the size.

Library production

Fragment library
After DNA fragmentation, library adaptors are added at both ends.

Mate paired end library
The DNA is fragmented. After adaptors have been added to the ends, the DNA is circularized. This circularized DNA is then cleaved, and after only fragments containing an adaptor have been purified, library adaptors are attached to each end.

Emulsion PCR

Library DNA is captured on beads via the adaptor sequence. The beads are encapsulated in a water-in-oil emulsion, forming a micro-reactor with a 1:1 ratio of beads to library DNA. PCR within the water-in-oil emulsion then clonally amplifies the library DNA on the beads to yield several million copies. After the reaction, the water-in-oil emulsion is broken down and the beads are recovered.

Sequencing

The beads are loaded individually into wells on a picotiter plate. One type of dNTP (dATP, dGTP, dCTP, or dTTP) is injected into each well. When a template-complementary dNTP is added, an elongation reaction mediated by DNA polymerase ensures that the dNTP is incorporated into the DNA chain, and pyrophosphoric acid is released. This acts as a substrate for the generation of ATP by sulfurylase. Luciferase then acts on this ATP and luciferin substrate in a luminescent reaction, and the signal intensity of this luminescence is detected by a CCD camera. Base sequences are determined from the presence of luminescence and its signal intensity.

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