Biomedical Engineering Reference
In-Depth Information
attached to the polyacrylamide matrix by acrydite modification [
6
,
7
] of the 5
0
end.
To perform the sequencing, the immobilized DNA is denatured, hybridized with a
primer and the complementary strand is elongated with deoxynucleotides that are
reversibly coupled to fluorescent groups. A scanning fluorescence microscope is
used to check if each type of deoxynucleotides has been incorporated or not, telling
the sequencing information of the complementary strand. After checking one type
of deoxynucleotides, the fluorescence is removed by cleaving the linker between
the fluorophore and the nucleotide. The cycle is repeated by adding a different type
of fluorescently labeled deoxynucleotide and scanning the gel. In this way, the
sequence of every target DNA molecule (represented by a PCR colony on the gel)
can be determined in parallel.
Based on the FISSEQ concept, but modifying the DNA amplification in gel
polonies by solid substrate “bridge amplification,” Solexa (now part of Illumina)
developed a DNA sequencing technology which is presently commercialized by
Illumina as the “Genome Analyzer System.”
11.1.3 Pyrosequencing
Pyrosequencing is a cyclic-array sequencing method based on detecting the
released pyrophosphate during incorporation of nucleotide triphosphate. The prin-
ciple of pyrosequencing [
8
] can be described as follows: (1) the target single
strand DNA molecules are fixed on solid surfaces, hybridized with a primer and
then incubated with DNA polymerase, ATP sulfurylase, firefly luciferase, and a
nucleotide-degrading enzyme apyrase; (2) the pyrophosphate (PPi) released during
the synthesis of DNA is converted to ATP by ATP sulfurylase and the concentration
of ATP is then sensed by the luciferase, which utilizes the energy of ATP to emit
light; (3) the amount of light can be estimated by a CCD (charge-coupled device)
camera; (4) unincorporated deoxynucleotides and the residue ATP are then
degraded by the nucleotide-degrading enzyme apyrase after each cycle; (5)
repeated cycles of deoxynucleotide addition are performed until the whole target
DNA is sequenced.
An example of the light signals resulting from the pyrosequencing reactions are
shown in Fig.
11.4
. The intensity of the signal at each cycle is proportional to
the number of nucleotides that are incorporated to each template during the cycle.
The technique has been further developed by a company named 454 Life Sciences
(now owned by Roche diagnostics) into a technology known as 454 Pyrosequencing.
For more details and other DNA sequencing technologies such as SOLiD
platform (Applied Biosystems; Foster City, CA, USA) and the HeliScope Single
Molecule Sequencer technology (Helicos; Cambridge, MA, USA), interested
readers may refer to references [
9
,
10
]. The remaining of this chapter will focus
on DNA sequencing approaches based on nanopore technology.
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