Biology Reference
In-Depth Information
filled with the capture beads, small enzyme-containing beads are added, and
the plate is placed into the sequencing instrument. The instrument can wash the
plate with A, C, G, and T nucleotides. The nucleotides are flowed sequentially in
four washes over the plate; when these nucleotides are incorporated into the
DNA strands, the enzymes on the enzyme-containing beads convert the chemi-
cals generated during the nucleotide incorporation into light in a chemilumines-
cent reaction similar to that of a firefly. The method is called
pyrosequencing
,
because it involves incubating the DNA-bearing beads with
Bacillus stearother-
mophilus
(
Bst
) DNA polymerase, single-stranded binding protein, ATP sulfury-
lase, and luciferase. When incorporation of a nucleotide occurs, pyrophosphate
is released, resulting in a burst of light that is detected by the recording device.
The signal strength is proportional to the number of nucleotides incorporated.
The 454 platform has difficulty in sequencing DNA regions with long strings
of the same bases because the length of such sequences is inferred from the
intensity of the signal and that can be a source of error. However, the 454 sys-
tem can generate
≈
400,000 reads with lengths of 200-1000 bp each, the longest
read lengths of the NextGen systems, but the most expensive.
Hayden (2009)
reported that this system sequences 1000 bp for US$0.05.
7.11.1.2 Illumina (or Solexa) Genome Analyzer
The Illumina analyzer, also known as the Solexa, uses adaptor-flanked DNA frag-
ments up to several hundred base pairs and bridge PCR to amplify these frag-
ments (
Bentley 2006, Fedurco et al. 2006
). First, the genomic DNA is fragmented,
and adaptors are ligated to both ends. The DNA is attached randomly to the
surface of the flow-cell channels that contain a dense lawn of primers. Next,
unlabeled nucleotides and enzyme are added to initiate bridge amplification. In
bridge PCR, both forward and reverse PCR primers are attached to a solid sub-
strate by a flexible linker so that all amplicons arising from a single template
molecule during amplification remain immobilized and attached in a single loca-
tion on the array. Bridge PCR relies on alternating cycles of extension with
Bst
polymerase and denaturation with formamide, resulting in clusters of
≈
1000
clonal amplicons. Several million clusters can be amplified at locations within
each of eight lanes on a single-flow cell, which means that eight different librar-
ies can be sequenced in parallel during a single run. Each cycle of sequence
analysis consists of single-base extension with a modified DNA polymerase and a
mixture of four nucleotides that are modified so that only a single base is incor-
porated in each cycle and one of four fluorescent labels is used to identify each
nucleotide. Read lengths are
≈
20-100 nt.
Hayden (2009)
reported that this plat-
form produces 1000 bp of sequence for US$0.002.
