Experimental Principles (Sequencing/Ion PGM)

Sample quality check

The concentration is measured with PicoGreen, and the size is checked by gel electrophoresis.

Library preparation

Amplicon sequencing using panels
After amplification of the target region by multiplex PCR, primer sequences in the amplicon are partially fragmented. An adaptor is then added to the amplicon DNA.

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 oil-in-water emulsion is broken down and the beads are collected.

Sequencing

The beads are loaded individually into tiny wells on a semiconductor microchip (Figure B). In the sequencing process, dNTPs (dTTP, dATP, dCTP, dGTP) are sequentially injected into the wells. When template-complementary dNTP is added, an elongation reaction mediated by DNA polymerase ensures that the dNTP is incorporated into the DNA chain, and hydrogen ions are released (Figure A). The charge carried by these ions changes the pH of the reagent, and this is detected by an ion sensor (Figure B). In this way, the Ion PGM reads bases by directly transforming chemical data into digital data (Figure C).

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