A Summary of DNA Sequencing, Technologies, Applications and Future Scope

PESU I/O: DNA Sequencing and Nanopore Technologies

Subgroup-6
1.1 Abstract: Deoxyribonucleic acid (DNA) is the
fundamental component of the genetic makeup of any
organism. DNA has the property of being unique to every
organism, even within a particular species. The basic
component of DNA that is responsible for the storage for
genetic code is termed a nucleotide. DNA Sequencing is the
process of identifying these nucleotides and the order in
which they are found in a sample of DNA. This paper aims
to describe in detail the past techniques used to sequence
DNA, the applications of DNA sequencing and the future
avenues that can be explored with this technique.
2.1 Introduction: DNA is an informative macromolecule in
the terms that it is used in transmission of genetic code and
characteristics from one generation to another. Owing to this
property, the technique of sequencing DNA has played a
pivotal role in deepening our understanding of evolution of
life on this planet and what order it followed. By comparing
genetic information of different species, it is easy to identify
where they overlap and where they diverge. The more
closely species are related on the phylogenetic tree, the
greater the amount of overlap in their DNA sequences. It can
be concluded that DNA sequencing plays a major role in the
present form of the study of evolutionary study of biology
among many other fields, and has proven to be an effective
and accurate method of scientific study.
3.1 Working principle: In one method of DNA sequencing DNA replication. The classical chain-termination method
called chain-termination sequencing, the DNA to be
sequenced, called the template DNA, is first prepared as a
single-stranded DNA. Next, a short oligonucleotide is
annealed, or joined, to the same position on each template
strand. The oligonucleotide acts as a primer for the synthesis elongation. These chain-terminating nucleotides lack a 3'-
of a new DNA strand that will be complementary to the
template DNA. This technique requires that four
nucleotide-specific reactions--one each for G, A, C, and T--
be performed on four identical samples of DNA. The four
sequencing reactions require the addition of all the
components necessary to synthesize and label new DNA,
including:
 A DNA template,
 A primer tagged with a radioactive material or a
photoemissive chemical,
 DNA polymerase,
 Four deoxynucleotides (G, A, C, T), and,
 One dideoxynucleotide(ddG, ddA, ddC, or ddT).
After the first deoxynucleotide is added to the growing
complementary sequence, DNA polymerase moves along the allow enough fragments to be produced while still
template and continues to add base after base. The strand
synthesis reaction continues until a dideoxynucleotide is
1 template DNA extension from the bound primer, the
added, blocking further elongation. This is because
dideoxynucleotides are missing a special group of molecules,
called a 3'-hydroxyl group, needed to form a connection with
the next nucleotide. Only a small amount of a
dideoxynucleotide is added to each reaction, allowing
different reactions to proceed for various lengths of time,
until, by chance, DNA polymerase inserts a
dideoxynucleotide , terminating the reaction. Therefore, the
result is a set of new chains, all of different lengths.
To read the newly generated sequence, the four reactions are
run side-by-side on a polyacrylamide sequencing gel. The
family of molecules generated in the presence of
ddATP are loaded into one lane of the gel and the other three
families, generated with ddCTP, ddGTP, and ddTTP, are
loaded into three adjacent lanes. After electrophoresis, the
DNA sequence can be read directly from the positions of the
bands in the gel.
4.1 Past methods used to sequence DNA:
In the past, the sequencing of DNA has been performed in
two primary methods that retain some relevance to this day.
They include
 Maxam-Gilbert sequencing, and
 Sanger-Coulson sequencing.
4.2 Sanger sequencing is a method of DNA sequencing ,
based on the selective incorporation of chain-terminating
dideoxynucleotides by DNA polymerase during in vitro
requires a single-stranded DNA template, a DNA primer, a
DNA polymerase, normal deoxynucleotidetriphosphates
(dNTPs), and modified di-deoxynucleotidetriphosphates
(ddNTPs), the latter of which terminate DNA strand
OH group required for the formation of a phosphodiester
bond between two nucleotides, causing DNA polymerase to
cease extension of DNA when a modified ddNTP is
incorporated. The ddNTPs may be radioactively or
fluorescently labelled for detection in automated sequencing
machines.
The DNA sample is divided into four separate sequencing
reactions, containing all four of the standard
deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the
DNA polymerase. To each reaction is added only one of the
four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP),
while the other added nucleotides are ordinary ones. The
dideoxynucleotide concentration should be approximately
100-fold lower than that of the corresponding
deoxynucleotide (e.g. 0.005mM ddTTP : 0.5mM dTTP) to
transcribing the complete sequence. Putting it in a more
sensible order, four separate reactions are needed in this
process to test all four ddNTPs. Following rounds of
resulting DNA fragments are heat denatured and separated
by size using gel electrophoresis. In the original publication
of 1977, the formation of base-paired loops of ssDNA was a
cause of serious difficulty in resolving bands at some
locations. This is frequently performed using a denaturing
polyacrylamide-urea gel with each of the four reactions run
in one of four individual lanes (lanes A, T, G, C). The DNA
bands may then be visualized by autoradiography or UV
light and the DNA sequence can be directly read off the X-
ray film or gel image.
4.3 Maxam–Gilbert sequencing is based on nucleobase-
specific partial chemical modification of DNA and
subsequent cleavage of the DNA backbone at sites adjacent
to the modified nucleotides.
Although Maxam and Gilbert published their chemical
sequencing method two years after Frederick Sanger and
Alan Coulson published their work on plus-minus
sequencing, Maxam–Gilbert sequencing rapidly became
more popular, since purified DNA could be used directly,
while the initial Sanger method required that each read start
be cloned for production of single-stranded DNA. However,
with the improvement of the chain-termination method (see
below), Maxam–Gilbert sequencing has fallen out of favour
due to its technical complexity prohibiting its use in standard
molecular biology kits, extensive use of hazardous chemicals,
and difficulties with scale-up.
Maxam–Gilbert sequencing requires radioactive labeling at
one 5′ end of the DNA fragment to be sequenced (typically
by a kinase reaction using gamma-32P ATP) and purification
of the DNA. Chemical treatment generates breaks at a small
proportion of one or two of the four nucleotide bases in each
of four reactions (G, A+G, C, C+T). For example, the
purines (A+G) are depurinated using formic acid, the
guanines (and to some extent the adenines) are methylated
by dimethyl sulfate, and the pyrimidines (C+T) are
hydrolysed using hydrazine. The addition of salt (sodium
chloride) to the hydrazine reaction inhibits the reaction of
thymine for the C-only reaction. The modified DNAs may
then be cleaved by hot piperidine; (CH2)5NH at the position
of the modified base. The concentration of the modifying
chemicals is controlled to introduce on average one
modification per DNA molecule. Thus a series of labeled
fragments is generated, from the radiolabeled end to the first
"cut" site in each molecule.
The fragments in the four reactions are electrophoresed side
by side in denaturing acrylamide gels for size separation. To
visualize the fragments, the gel is exposed to X-ray film for
autoradiography, yielding a series of dark bands each
showing the location of identical radiolabeled DNA
molecules. From presence and absence of certain fragments
the sequence may be inferred.
2 and Eskimos indicated that they could survive in high
5.1 Applications of DNA sequencing:
General applications of DNA sequencing include usage in
fields like
 Metagenomics: Study of genetic material recovered
directly from environmental samples
 Medicine: Genetic disorder identification, gene
therapy
 Forensic science: Crime investigation, paternity
testing, DNA fingerprinting
 Molecular biology: Protein synthesis, physiological
analysis, drug response mechanisms, phenotype
identification
 Evolution studies: Timeline formation and so on.
5.1.1 Specific Application: Evolution of Organisms
The patterns and sequences within DNA are analyzed to
arrive at a logical hierarchy related to the evolution of
organisms.
The components of study include primarily
1. The phylogenetic tree,
2. Evolutionary clock theory,
3. Extinction,
4. Morphology of organisms,
5. Habitat of organisms, and,
6. Behavior of organisms
The phylogenetic tree: More overlap within the DNA
samples of 2 organisms imply that they are more closely
related than those organisms whose samples show less
overlap.
Evolutionary clock: The genetic samples help determine the
era during which 2 species diverged from a common ancestor.
This is measured on a relative scale using known data, as
absolute time cannot be known from just a sequencing
routine. Larger differences in the DNA indicate larger delta
in the eras of existence. This method of studying this field, is
of course, limited in utility by virtue of a limited population
size, protein function changes, change in mechanism of
natural selection, species-specific anomalies, and changing
periods between generations.
Extinction: Divergence offers clues about mass extinctions,
as well as single extinction and the probable cause of death--
exempli gratia, the probable presence of a mutated gene in
mammoths.
Morphology of organisms: Explaining by way of example,
it is known that Neanderthal DNA differs from modern
human DNA by 0.3%-- this includes the MC1R gene, which
provides instructions to make a protein called the
melanocortin 1 receptor—responsible for skin and hair
colors. The Neanderthal human was believed to have reddish
hair and light skin—mating with modern humans resulted in
individuals who now inhabit Europe and Asia.
Habitat of organisms: Explaining by way of example, the
Denisovans were a class of humans who inhabited present
Siberia. Similarities in their DNA to Aborigines, Tibetians
altitude conditions with frigid temperatures. This can also
serve as explanation to the mammoths’ extinction due to
inbreeding as they tended to live in isolated islands.
Behavior of organisms: Changes in the FOXP2 gene led to
humans using language and expressions, while apes that lack
this mutation cannot use language. The ‘fruitless’ gene of
Drosophila melanogaster determines mating and courtship
behavior. Food, foraging and psychological behavior also
works this way to an extent.
5.1.2 Past experiments and hypotheses
The Genographic Project: In 2005, NGS and IBM launched
the Genographic Project to retrace the earliest human
migrations using DNA sequencing, using samples donated
by people all over the world. This project provided valuable
insight into the origin as well as the later migrations that
brought humans to occupy all corners of the globe. By
calculating the pattern of genetic diversity in different
populations that arises due to mutations, the age and ancestry
of people living in different regions can be deduced. From
this work, it is now known that humans originated in Africa
and left the continent about 60,000 years ago to populate the
planet. This project also revealed that the Y chromosome is
passed down virtually unchanged from father to son and that
DNA in the cell’s mitochondria is passed down from the
mother.
Molecular clock hypothesis: The molecular clock
hypothesis states that DNA and protein sequences evolve at a
rate that is relatively constant over time and among different
organisms. Therefore, if the hypothesis is true, this is an
extremely useful method for estimation of evolutionary
timelines.
Bacterial classification: DNA sequencing also facilitated the A-I.2 References
identification of pathogens such as Heliobacter pylori and
the Mycobacterium species. Pyrosequencing of 16S vRNA
gene was also used to develop a molecular gram stain in
order to rapidly classify bacteria using molecular methods.
6.1 Future applications and scope
 Some organisms which no longer exist or certain
traits associated with them could be brought back to
life.
 Exaptation is the repurposing of a certain sequence
for a different function. This happens in non-coding
sequences, or exons. It has happened a lot in the last
400 million years during evolution and can be
exploited in the future.
 Epigenetics is the blocking or accessing of certain
genes. Blocking by DNA methylation or even
changing the sequence altogether can be done for
specific purposes.
3
7.1 Conclusion and afterthoughts
From the above findings, it can be argued with reasonable
validity that DNA sequencing as a field has a lot of future
potential and continued research in this field will serve to
deepen our understanding of ourselves and what led us to
existence. It can also be argued with reasonable validity that
the practice of gene manipulation can be beneficial to
medicine and to victims of forced mutations by way of
nuclear disasters and the like.
Appendix-I
A-I.1: Conventional DNA sequencing
 Sanger F; Coulson AR (May 1975). "A rapid
method for determining sequences in DNA by
primed synthesis with DNA polymerase"
 Maxam AM, Gilbert W (February 1977). "A new
method for sequencing DNA". Proc. Natl. Acad. Sci.
U.S.A.
 Sanger F, Coulson AR (May 1975). "A rapid
method for determining sequences in DNA by
primed synthesis with DNA polymerase". J. Mol.
Biol. 94
 King, Turi E.; Jobling, Mark A. (2009). "What's in a
name? Y chromosomes, surnames and the genetic
genealogy revolution". Trends in Genetics. 25
 Wells, Spencer (2013). "The Genographic Project
and the Rise of Citizen Science". Southern
California Genealogical Society (SCGS)
 Harry, Debra and Le'a Malia Kanehe. "Genetic
Research: Collecting Blood to Preserve Culture?"
Cultural Survival, 29.4 (Winter 2005)