Page 1344 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPTEr 96 Molecular Methods 1305

structural variants in DNA. In addition, an important recent the cost of the all-electronic detection system is far less than other
application of next-generation sequencing (NGS) will be dis- sequencing instruments.
cussed, circulating cell-free DNA (cfDNA). This technology has Long read sequencing—single-molecule sequencing and
already revolutionized pregnancy screening for common nanopores. A true single-molecule sequencing method has been
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chromosomal disorders and may be adapted to screening for developed by Pacific Biosciences (Menlo Park, CA). In their
low levels of solid tumor DNA in blood. method DNA polymerase is immobilized in a femtoliter-sized
Sequencing by synthesis (Illumina). The most widely used well with special optical properties called the zero-mode wave-
system for NGS involves highly parallel imaging of single base guide (ZMW) (see Fig. 96.6C). The small size of the ZMW “hole”
addition on “clusters” of identical DNA molecules (see Fig. 96.6A). prevents 600-nm wavelength laser light from passing entirely
This technology has been commercialized by Illumina Inc. (San through, and only the bottom 30 nm where the polymerase is
Diego, CA). In their approach, DNA is ligated to primers that bound is subject to the fluorescence excitation. Labeled nucleotides
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allow it to be amplified on a surface called a “flow cell.” The are flowed into the chamber and complementary bases encounter-
DNA templates attach to the flow cell surface by hybridization ing the DNA polymerase are incorporated into the growing DNA
to specific primers complementary to that used to prepare the chain. During incorporation, the DNA polymerase holds the
DNA library. Solid-phase bridge amplification creates many nucleotide for tens of milliseconds, orders of magnitude longer
identical copies of each single template molecule in a localized than the average diffusing nucleotide. The transient light emission
cluster. The density of the clusters is extremely high, and the is then detected and the identity of the incorporated base recorded.
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specialized imaging system allows resolution of more than 10 Nucleotides with a fluorescent dye attached to the phosphate
clusters per flow cell. DNA sequence is determined in the flow group of the nucleotide are cleaved when the nucleotide is
cell by sequential addition of fluorescent-tagged nucleotides. A incorporated into the DNA strand. The label diffuses away, leaving
single-labeled nucleotide is added to nascent DNA in each the DNA ready for the addition of the next base. The polymerase
sequencing cycle. Each nucleotide has a different fluorophor so incorporates multiple bases per second, so the sequencing process
that all four are added to the mix in each cycle of base extension. is very fast with real-time observation of DNA synthesis.
The nucleotide label also terminates polymerization (i.e., only Engineered nanopores are being evaluated for the detection
a single base is added at each cycle), and the identity of the of a broad array of biomolecules, including proteins and nucleic
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incorporated nucleotide is determined by the fluorescent emission acids. There are many potential variations in pore, sample
spectrum and intensity. Because the bases act as terminators, preparation, and detection, which have given rise to active
homopolymeric segments can be resolved relatively accurately. academic and commercial research programs. In one effective
After each deoxynucleotide (dNTP) incorporation, the fluorescent version, a protein channel (e.g., Mycobacterium smegmatis porin
dye is enzymatically cleaved to allow incorporation of the next A, MspA) through which DNA may pass is held in a partition
nucleotide. This class of instruments is capable of sequencing (membrane or other solid) that separates solutions containing
complete human genomes in a single run. charged ions. An applied voltage leading to movement of ions
Electrochemical sequencing. Electrochemical sequencing through the protein pore produces a measurable electrical current.
(IonTorrent/Proton; ThermoFisher Scientific, Waltham, MA) is DNA passing through the channel causes partial reduction of
distinctly different from systems that image fluorescent-tagged the current with the four DNA bases affecting the current by
nucleotides because the sensor is a microelectronic device (see differing and characteristic amounts (see Fig. 96.6D). The chang-
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Fig. 96.6A). DNA is first fragmented and then ligated to adapters, ing electrical current is then used to infer the order of bases in
and the adaptor-ligated libraries are clonally amplified by emulsion the DNA molecule. Oxford Nanopore Technologies (Oxford,
PCR onto beads. Individual beads are then loaded into single-sensor UK) released the first commercial devices based on this technology,
wells. Nucleotides are provided in a stepwise fashion with incor- and the uses are under active investigation. One of the most
poration increasing the length of the sequencing primer by one important strengths of this method is the very long single-
base when there is a complementary base on the template strand. molecule reads that can be obtained. High read depth can partially
Sequence determination relies on primer extension. Nucleotide overcome problems with sequence accuracy (per base error rates
incorporation into a nascent DNA strand by DNA polymerase of 10–30%). The fast run times and simple protocols for sample
results in hydrolysis of the nucleotide triphosphate. Hydrolysis preparation have allowed an early stage instrument to be used
causes production of a hydrogen ion for each nucleotide incor- to sequence Ebola virus on site in Africa. Because of the speed
porated. The small shift in the pH of the surrounding solution of analysis, “bedside” medical applications could be feasible
is proportional to the number of nucleotides incorporated, which (especially when base error rate is of less importance for the
is then detected by the sensor on the bottom of each well, converted intended use) and appears especially promising in the arena of
to a voltage, and digitized by off-chip electronics. The chip is precision diagnosis of infectious diseases.
automatically washed, and the cycle is repeated with the next
nucleotide. Because of the small size of the wells, diffusion into BIOINFORMATICS
and out of the well is very fast. The sensor layer is composed of
tantalum oxide, which is sensitive to proton concentration Clinical Bioinformatics has emerged as a fundamental discipline
(essentially pH) allowing rapid detection of the voltage transients in laboratory medicine as enormous DNA sequence, transcrip-
that follow base incorporation in each well individually. The voltage tomics, and variant databases have been aggregated from indi-
change is roughly proportion to the length of a run of the same vidual laboratories and international genomics projects. The
nucleotide in the DNA so that short homopolymers can be growing number of genetic loci that are already known to be
accurately called. Chip fabrication and design are very similar to important in immunological disease renders it critical that
methods used for microelectronic devices. This suggests that the diagnostic laboratories make maximal use of automated processes
method will have good potential to scale up to increase the number in sample management, data acquisition, data analysis, and
of molecules processed in parallel. Lacking optical components, reporting. Laboratories using NGS instruments must establish