DNA

SEQUENCING
Mikel Herskovits 4 °D
Index
1. INTRODUCTION................................................................................................................................... 2
2. PROPERTIES OF DNA............................................................................................................................ 2
3. DNA REPLICATION............................................................................................................................... 3
4. SANGER SEQUENCING.......................................................................................................................... 4
4.1. REPLICATION.............................................................................................................................................5
4.2. BASE DETECTION....................................................................................................................................5
4.3. DATA PROCESSING..................................................................................................................................5
5. NEXT-GENERATION SEQUENCING......................................................................................................... 6
6. CONCLUSION....................................................................................................................................... 6
7. BIBLIOGRAPHY.................................................................................................................................... 6
 1. Introduction
DNA sequencing is a process used to determine the order in which the bases of
nucleotides are arranged in a strand of DNA. In 1990, the Human Genome Project began its
operations and its aim was to map out the DNA of a homo sapiens; after over a decade of
international efforts, the report was complete on April 2003. Thirteen years to break down
the genome of a human being was revolutionary for the time, nevertheless, thanks to new
methods and technologies, DNA sequencing is cheaper and less time consuming than it was
back then. Despite it being far easier than it used to be, DNA sequencing is still a very
complex and rigorous process with lots of intricacies.

2. Properties of DNA
In order to understand how DNA Sequencing works, we first have to be familiarized
with DNA itself. In consonance with the US National Library of Medicine (2020),
deoxyribonucleic acid – or DNA for short – is a molecule composed of two chains that coil
around each other in the shape of a double helix; they carry the genetic material which
instructs the organism how to function, grow and reproduce. The smallest unit of DNA is a
nucleotide, which looks like the following:

The nucleotide is made up of three components: the deoxyribose – which acts as the
structure of DNA, the phosphate group – which mends nucleotides together, and the
nuclear base – which contain the codes that make up the genetic material; for the purposes
of DNA sequencing, we will focus on the latter. A nuclear base contains one letter out of
four possibilities: A for adenine, T for thymine, C for cytosine and G for guanine. But because
DNA is in the form of a double helix, the second strand must also contain its own
nucleotides arranged in an antiparallel manner; for compatibility reasons, A bases only bond
with T, and C bases with G. The order of these letters indicates what the genetic material is;
therefore, sequencing DNA actually refers to deciphering these letters and their order to
find the codes of the DNA.
 3. DNA Replication
DNA replication is an essential process in the cell cycle during which a cell creates a
copy of its genetic material; moreover, it also plays a key role when DNA sequencing is
performed. For the sake of simplicity, it has been separated into six fundamental steps:

1. It begins with an enzyme called helicase, which pulls the two strands of the DNA in
its double helix apart, identifying the DNA double helix:

2. Once it has set its target, the helicase will separate the strands and topoisomerase –
another enzyme – will bind to them and keep the strands at a distance from each
other. This space allows for the primer to come in and act as a foundation on which
the DNA polymerase – an enzyme that synthesizes new DNA nucleotides according
to the information found on the respective nuclear bases in a 5’ to 3’ order.

3. Then, the helicase will continue to pull the strands apart. Because the DNA
polymerase synthesizes the new strands in a 5’ to 3’ order, one of the strands will
have to continuously introduce a primer and a DNA polymerase to fill in the gap. As a
consequence, replicating the second strand takes more time than the first;
therefore, the faster is called the leading strand and the slower is the lagging one.

4. By the time the helicase has completely separated the strands, we will be left with
two DNA structures. The first is the leading strand, which will be continuous and only
has one primer, whereas the lagging strand has several. Because primers don’t
 correspond with the genetic material, a new enzyme – called ligase – will be
introduced to replace the primers and join the gaps between the Okazaki fragments.

5. Once the ligase has replaced the primers and joined the DNA polymerase fragments,
the topoisomerase will begin to detach from the stands – allowing them to recoil
into a double helix.

6. Finally, the helicase will separate itself from the strands - these will continue to coil.
As stated before, the leading strand will finish before the lagging one because it got
an early start on step 5. Eventually, both pairs of strands will have recoiled into the
double helix and DNA replication will be complete.

4. Sanger Sequencing
But in order to understand modern day DNA sequencing, we first have to go back to
the 1977, when the British biochemist Frederick Sanger developed Sanger sequencing – also
known as the chain termination method – that would go on to be used for over two
decades. According to the Human Genome Research Institute (2015), this method can be
used to determine the sequence of up to 900 base pairs and it was this technology that
allowed scientists in the 90s to map out the DNA of a human. The Sanger sequencing
procedure can be separated into three steps: replication, base detection and data
processing.
4.1. Replication
Sanger sequencing begins by selecting the section of interest and heating it so that
the strands separate. Then, just like in replication, a primer is set, and the substance is
cooled so that it can bind to the strand. Next, the strand is heated again, allowing the DNA
polymerase to begin to replicate the DNA using the primer as a starting point. Once the
section of interest had been replicated, a dideoxy ribose – a nucleotide without the 3’
hydroxyl group – is introduced which acted as a stop button and effectively ceases the
replication. At this point, no further deoxyribose can be added because there is no 3’ to
connect to; thus, the section ends with a dideoxy ribose.

4.2. Base Detection

This procedure is repeated several times in units known as cycles; by the time
they’re done, it’s almost guaranteed that the replication will have ended with a dideoxy
ribose at least once in each of the positions of the target DNA. All of these strands are then
put through a process capillary gel electrophoresis, which filters the fragments via a tube
and passes them through a laser at the end that illuminates them. Each base type is dyed in
a different color; generally, adenine is green, guanine is pink, cytosine is blue, and thymine is
orange. Because each base type is colored differently, when the laser illuminates the strand,
they will emit a frequency accordingly and it will be detected by a light sensor.

Capillary Gel Electrophoresis – University of New South Wales (2018)

4.3. Data Processing

Finally, the results are processed by a computer and they will be represented in the
form of a chromatogram – a visible record of the light intensities that were emitted; from
this graph, professionals are able to read from the peaks which base types correspond in
each position. With that, the Sanger sequencing will be complete as the base types of the
section of interest will have been discovered.
 Chromatogram – Khan Academy (2016)

5. Next-generation Sequencing
While Sanger sequencing produces high quality results, it’s a very expensive and
tedious method that proves inefficient when it comes to large scale projects; for these
cases, there are newer, cheaper, faster and therefore more adequate techniques.

In consonance with the European Bioinformatics Institute, while there’s a wide

variety of next-generation procedures to sequence DNA, they all share the concept of
running thousands of small-scale Sanger sequences simultaneously. As a consequence,
sequencing DNA has become faster – because the reactions are taking place at the same
time, and cheaper – due to developments in the equipment. In fact, the Human Genome
Project completed in 2001 cost almost $100 million, whereas it nowadays costs just over
$1000.

6. Conclusion
To conclude this report, DNA Sequencing is a very useful genetic procedure that is
heavily tied to replication. It was invented in the late 70s, when Frederik Sanger devised a
method to reveal the base types of a specific section of DNA, and his technique has been
improved and built upon ever since. While we don’t know what the future holds, and recent
pandemics have been tormenting our species, we hope that DNA sequencing is used to
further our developments in fields of science and medicine that will help us prepare for
future threats.

7. Bibliography
Rafael N Añez Regidor, “Molecular Biology: Replication”, YouTube, Published On: 9/3/20, Accessed
On: 12/3/20, https://www.youtube.com/watch?v=sEBAPiCyzo8&feature=emb_title

Khan Academy, “DNA Sequencing”, Published On: 2016, Accessed On: 14/3/20,
https://www.khanacademy.org/science/high-school-biology/hs-molecular-genetics/hs-
biotechnology/a/dna-sequencing

University of North Texas, “DNA Sequencing Core Facility”, Department fo Biological Sciences,
Published On: Unknown, Accessed On: 16/3/20,
https://www.biol.unt.edu/~jajohnson/DNA_sequencing_process

National Human Genome Research Institute, “DNA Sequencing Fact Sheet”, National Institutes of
Health, Published On: 18/12/15, Accessed On: 14/3/20, https://www.genome.gov/about-
genomics/fact-sheets/DNA-Sequencing-Fact-Sheet

US National Library of Medicine, “What is DNA”, Published On: Genetics Home Reference, Published
On: 17/3/20, Accessed On: 16/3/20, https://ghr.nlm.nih.gov/primer/basics/dna

University of New South Wales, “Guide to Sanger Sequencing”, Ramaciotti Centre for Genomics,
Published On: 27/9/18, Accessed On: 16/3/20, https://www.ramaciotti.unsw.edu.au/sites/default/files/2019-
04/RAMAC_Sanger_Sequencing_Service_Guide_2019_v1.0.pdf
European Bioinformatics Institute, “What is Next Generation Sequencing?”, EBI, Published On: 2018,
Accessed On: 17/3/20, https://www.ebi.ac.uk/training/online/course/ebi-next-generation-
sequencing-practical-course/what-you-will-learn/what-next-generation-dna-