Sanger Sequencing and 

Next-­
Generation Gene Sequencing: 23
Basic Principles and Applications
in Pathology

DNA sequencing means the determination of the

order of sequence of the base pair in DNA.

23.1 Sanger Sequencing

Sanger sequencing is named under Sanger F. This is

the widely used commercial DNA sequencing [1].

Basic Principle  In Sanger sequencing tech-

nique, 2′,3′-dideoxynucleotides are used for
DNA synthesis. In the absence of 3′-hydroxyl
group in 2′,3′-dideoxynucleotides, DNA can-
not be synthesized further as no phosphodiester
bond can be formed with the next dNTP, and the
chain terminates (Fig. 23.1). The four DDNTPs
(dideoxynucleotides phosphates such as ddATP,
ddTTP, ddCTP, and ddGTP) are labelled by dif-
ferent fluorochrome dyes so that they are recog- Fig. 23.1  Schematic diagram showing the principle of
nized by laser beam. Each fluorescent-labelled Sanger sequencing technique. Here 2′,3′-dideoxynucleo-
terminated fragment of DNA is recorded and tides are used for DNA synthesis, and in the absence of
from this data DNA sequence is assessed. 3′-hydroxyl group, the DNA chain is terminated. The four
DDNTPs are labelled by different fluorochrome dyes so
that they are recognized by laser beam, and from this data
Reagents needed: DNA sequence is assessed

• Primers: Small piece of single-stranded DNA

is used. • ddNTP (fluorescent labelled): All four ddNTPs
• DNA template: This chain is sequenced. such as ddATP, ddTTP, ddCTP and ddGTP
• DNA polymerase enzyme. are labelled with different fluorochrome dyes.
• dNTPs (dATP, dTTP, dCTP, dGTP). These are chain-terminating nucleotides.

© Springer Nature Singapore Pte Ltd. 2018 227

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228 23  Sanger Sequencing and Next-Generation Gene Sequencing
Main steps:
• Mix the above reagents, and denature the
complementary strands of DNA template by
heating at 96 °C.
• Lower the temperature to 50 °C for annealing
the primer with DNA template.
• Raise temperature to 60 °C for the elongation of
DNA with the help of DNA polymerase.  The
DNA chain elongates till it incorporates
fluorescent-­labelled ddNTP.  At that point the
DNA chain elongation is terminated. The DNA
chain is terminated with various lengths bearing
the terminal-labelled ddNTP (fluorochrome-­
labelled A, T, C and G).
• The PCR cycle of DNA is repeated several
times to have all the base position of the DNA
template. At the end, variable lengths of DNA
chains with terminal unique fluorescence
labelled A, T, C and G are obtained.
• DNA chains undergone capillary gel electro-
phoresis, and then the coloured emission spec-
trum of all these fluorochrome-labelled DNAs
is recorded by capillary gel electrophoresis
(Fig. 23.2). With the help of a suitable software
from the data of the terminal bases, the exact
sequence of the bases in the DNA is generated.
Fig. 23.2  Schematic diagram showing the capillary gel
electrophoresis of different fragments of PCR products
23.2 Next-Generation Sequencing
Next-generation sequencing (NGS) is the newer
gene sequencing technology that rapidly per-
forms sequencing from abundant small fragments
of DNA [2]. NGS is also called as high-­throughput
sequencing.
There are several categories of NGS:
1. Pyrosequencing
2. Microelectrophoretic methods
3. Hybridization sequencing
4. Real-time observation of single molecules
1. Pyrosequencing [3]: The pyrosequencing tech-
nology was the first available NGS system
in the market. Pyrosequencing works on the
principle of “sequencing of sequencing”. In
this technique fixed amount of inorganic pyro-
phosphate (PPi) is released whenever a nucle-
otide is incorporated in polymerization reaction
by DNA polymerase enzyme (Fig.  23.3). The
released PPi initiates a chain of reaction that
liberates light energy and is detected by colour-
charged device camera. The DNA sequence is
assessed from the pyrogram that is generated
during each nucleotide incorporation.
Advantages:
• Compared to Sanger sequencing, it is very
fast.
• Much cheaper than Sanger sequencing.
• DNA sequencing of the different samples can
be determined by the same run.
Limitation:
• Error-prone in case of the sequencing of long
homopolymers
2. Microelectrophoretic method: In microelec-
trophoretic method microfabricated technical
device is used for the electrophoretic separa-
tion of the DNA fragments [4]. Here an intri-
cate ultradense channel arrays are made that
help in micron precision. This technique has
23.2  Next-Generation Sequencing 229

Fig. 23.3 Schematic
diagram showing the
basic principle of
pyrosequencing
technique. Here fixed
amount of inorganic
pyrophosphate (PPi) is
released whenever a
nucleotide is
incorporated in
polymerization reaction
by DNA polymerase
enzyme. The released
PPi initiates a chain of
reaction that liberates
light energy and is
detected by colour-­
charged device camera.
The DNA sequence is
assessed from the
pyrogram that is
generated during each
nucleotide incorporation

integrated sample processing, DNA amplifi-
cation, isolation and concentration of DNA
fragments and sequencing.
Advantages:
• Very fast technique
• Minimal reagent consumption
• Good quality optical property
3. Hybridization sequencing (Fig.  23.4): In this
technique, numerous oligonucleotide probes are
used to hybridize with the target DNA. The com-
plementary probes are hybridized with DNA,
and therefore the sequence of bases of DNA is
obtained. Finally the complete DNA sequence is
decided by assembling the overall information
from the multiple hybridization tests.
There are two ways to hybridize: (a) The DNA
is immobilized on a membrane, and then various
small oligonucleotide probes are used for hybrid-
ization. (b) Microfabricated tilling array contains
more than 6,000,000 distinct probes, and the
genomic DNA to be sequenced is hybridized to
determine the complete base sequence of the
entire DNA [5].
Advantages:
• Fast
• High throughput
230 23  Sanger Sequencing and Next-Generation Gene Sequencing

• Clinical decision support: The demonstration

of complete mutation data of a disease may
help in the identification of the driver muta-
tion. Once the driver mutation is identified, we
can plan a therapy of the individual patient on
the basis of it such as erlotinib EGFR muta-
tion and vemurafenib in BRAF mutational
melanoma.
• Study of cancer genome for diagnosis, classi-
fication and prognosis: NGS is able to provide
the complete data of the individual cancers.
This data may be helpful in the diagnosis,
classification and prognosis aspects of the
disease.

23.2.2 Limitations

• Cost: NGS is very costly and needs adequate

Fig. 23.4  Schematic diagram showing the basic princi-
infrastructure, experience, storage capacity
ple of hybridization sequencing. In this technique, numer-
and computational capacity.
ous oligonucleotide probes are used to hybridize with the
target DNA, and the sequence of bases of DNA is • Intratumor heterogeneity: There may be varia-
obtained. Finally the complete DNA sequence is obtained tion of mutations in a particular type of tumor
by assembling the overall information from the multiple
in different patients (intertumoral heterogene-
hybridization tests
ity) or different mutational changes in a same
tumor at different sites (intratumor heteroge-
4. Real-time observation of single molecules: In
neity). This is a complicated issue, and there-
real-time sequencing, DNA chain is passed
fore the response of therapy may vary in a
through a nanopore. This is an alpha-­
particular tumor.
haemolysin pore covalently bound with cyclo-
dextrin. The different base pair of DNA
generates fluctuation of electrical signal dur-
ing the passage through the nanopore, and the
23.3 Comparison of Sanger
exact DNA sequence is assessed by measur-
Sequencing and NGS
ing the fluctuation of pore conductance [6].
The differences of the above two techniques are
Advantages: highlighted in Table 23.1.

• There is no need of DNA amplification or

Table 23.1  Comparison of Sanger sequencing and Next-
cloning. generation sequencing
• Read length of the technique is much longer
Sanger Next-generation
than other techniques. Features sequencing sequencing
Speed Very slow Fast
Knowledge of The prior Unselective and
23.2.1 Scope of NGS complete knowledge of no need of
genomic data the loci of the knowledge of
gene is needed loci of gene
• Identifies broader spectrum of mutational
Detailed Only base Detailed
changes: In addition to base sequence of DNA, molecular sequences mutational
NGS detects all novel mutational changes mutational possible changes can be
such as small insertions and deletions. change demonstrated
References
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