View larger version (41K):
[in this window]
[in a new window]
|
Fig. 2 Outline of the 454 and polony sequencing process. Both systems first
fragment the genomic DNA (Step 1) and then use a process of in vitro
cloning followed by amplification. The 454 process is shown on the left and
Polony sequencing is shown on the right. In the 454 protocol, the linkers are
ligated onto the ends of the DNA (Step 2a). Polony sequencing involves
circularization followed by linearization and the addition of linkers to
generate two fragments with a spacer between them and linkers at the end (Step
2B). Both processes then attach the in vitro clones to beads and
carry out PCR in an emulsion mixture to generate beads with many clonal copies
of the target fragments (Step 3a/3b). For the sequencing step, the beads must
be immobilized in a single layer to allow imaging in an environment that
enables the reaction reagents to be flowed across them. In the case of 454
sequencing, a picotiter plate is used, in which most cells will contain a
single bead (Step 4a). The polony method immobilizes the beads in an
acrylamide matrix in a dense monolayer (Step 4b). The methods are very similar
up until the point of the sequencing reaction; in the case of 454 sequencing,
a DNA synthesis reaction from a single sequencing primer is carried out. Bases
are flowed across the picotiter plate one at a time and incorporation is
detected by the release of light (Step 5a). The polony method uses ligation to
anchor primers, which can be annealed in one of four positions. In each cycle,
a population of degenerate nonomers, which have been fluorescently labeled, is
added to the monolayer, and only complimentary oligos will anneal and ligate
to the anchor primer.
|
