Page 1342 - Clinical Immunology_ Principles and Practice ( PDFDrive )

CHaPTEr 96 Molecular Methods 1303

expression analysis, cDNA “target” derived from the tissue or
cell source is labeled with fluorophores and used to determine Sanger Sequencing
complementary binding by massively parallel hybridization to The direct determination of a DNA sequence is fundamental to
the probes fixed on the array. The ability to massively sequence mutation identification (Fig. 96.5). The oldest method still in
RNA (see below) from diverse biological samples has reduced common use (called Sanger sequencing) depends on termination
use of arrays for research. RNA measurement, either by array of DNA synthesis by chemically modified nucleotides (dideoxy-
or sequencing, has been slow to enter clinical practice. A major NTPs [ddNTPs]). A single-stranded DNA (ssDNA) template is
challenge lies in the variation between studies in sample selection, hybridized to a primer that can recognize a short known segment
cell composition of samples, analysis platform, and statistical of DNA. The ssDNA is usually produced by DNA amplification.
methods. Establishing criteria for analytical validity, clinical The primer acts as a starting point for DNA synthesis by a DNA
validity, and clinical utility are long-term challenges for use of polymerase that is added to the reaction. Four separate reactions
gene expression biomarkers. are set up, each “spiked” with one of the four possible ddNTPs
(ddATP, ddTTP, ddCTP, or ddGTP). As synthesis proceeds, some
Detection of Disease Causing Mutations—Point of the strands incorporate a ddNTP, and no further extension
Mutations, Insertions/Deletions, and Structural Variants can take place. In each reaction, a family of molecules is syn-
Methods for the detection of single-base substitutions in DNA thesized; their unit lengths are determined by whether the reaction
27
are central to clinical genetic diagnostics. Although comple- had been terminated by incorporation of a specific ddNTP at a
mentary DNA (cDNA) synthesized from mRNA can be used, given position. Either the primer or the nucleotides may be
analysis of genomic DNA is far more common. Many different labeled. Automated instrumentation for DNA sequencing using
methods for mutation detection have been described, but virtually laser scanning of fluorophore-labeled reactions is standard. By
all clinical diagnostic laboratories now approach an unknown using four different fluorescent labels the reaction products from
mutation with DNA sequencing. When Sanger sequencing is one sample can be analyzed together, with many samples processed
used, the first step is to design PCR primer sets that will allow in parallel using renewable capillary electrophoresis arrays.
amplification of each exon. The availability of the reference human
genome sequence makes determination of gene structure and Next-Generation DNA Sequencing
amplimer design for genomic DNA straightforward. Any mutation New technologies that have dramatically increased the throughput
that directly affects amplification, such as deletion or mutation and reduced the cost of DNA sequencing have been rapidly
in the amplimer sequence, can produce misleading results. It is adopted in research and diagnostic laboratories. Unlike Sanger
always desirable to have parental specimens available to serve sequencing, which produces sequence data by controlled termina-
as a reference for proving biallelic inheritance in the affected tion of the polymerase, the new techniques derive the sequence
offspring. If there is a clear clinical diagnosis and only a single as nucleotides are sequentially added by the polymerase or
gene likely to be involved, then automated fluorescent sequencing interrogate the sequence by flow through nanopores. Although
is still the preferred method. For disorders with highly complex multiple chemistries and detection systems have been developed
clinical and laboratory phenotypes, like primary immune deficien- into sequencing instruments (Fig. 96.6), this discussion will review
cies, the problem of locus heterogeneity is very severe. It is now methods that have been adopted into clinically validated assays
routine practice to solve this problem by sequencing the complete and also briefly describe two methods that, to date, have only
exome or the complete genome in individual patients by using been used in research but that illustrate the potential for very
the next-generation technologies described below. long reads. Such methods may have a role in better defining

Universal
sequencing primer
ssDNA

Dideoxy
termination
reaction

T C G A
ddA
ddG Denaturing
ddG PAGE
ddT
ddT
ddC
ddT
ddC
ddG
ddA
ddA
ddG
FIG 96.5 DNA Sequencing by Dideoxynucleotide (ddNTP) Chain Termination (Sanger). Copying
of the DNA by the polymerase is terminated at specific positions when a ddNTP is incorporated. The
ddNTP is mixed with deoxynucleotides (dNTPs) so that in each reaction, only some new strands
terminate, whereas others continue through to the next complementary nucleotide. The sequencing
products can be visualized by autoradiography or by laser scanning in an automated sequencer.