Understanding the 454 Method

A DNA strand is prepared by cutting the sample into small fragmented pieces (Fig 1). Attached to the ends of these fragments are oligonucleotide adaptors (Fig 2). These allow the fragments to individually attach to primer-coated beads. The goal is to have one fragment per bead. Amplification essentially copies fragments on each bead (Fig 3). Beads are then filtered, ridding of unattached DNA fragments. For sequencing, a single bead is placed into a picotiter-volume well on a plate accompanied with an enzyme bead to be incubated. Nucleotide bases are released in waves. A light signal is generated when each base is incorporated. The intensity of light is proportional to the number of repeated nucleotides of the same type.

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