DNA SEQUENCING IN GENETICS, BIOLOGY AND MEDICINE

BY

EHIOROBO FAVOUR
AST/2372300577

A SEMINAR WORK SUBMITTED TO THE DEPARTMENT OF

BIOCHEMISTRY, SCHOOL OF APPLIED SCIENCE AND
TECHNOLOGY, AUCHI POLYTECHNIC, AUCHI
IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE
AWARD OF HIGHER NATIIONAL DIPLOMA (HND) IN
BIOCHEMISTRY.

NOVEMBER, 2024

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 ABSTRACT
DNA sequencing has revolutionized our understanding of genetics, enabling
researchers and clinicians to decode the genetic information embedded in organisms.
This seminar will delve into the various DNA sequencing technologies the profound
implications of these advancements in genomics, personalized medicine, and
biotechnology. DNA sequencing stands at the forefront of modern biology and
medicine, with its continued advancement promising to unlock new frontiers in our
understanding of genetics. This seminar will highlight the transformative potential of
DNA sequencing technologies, emphasizing their implications for health care,
research, and society.

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 OUTLINE

Title Page - - - - - i

Abstract - - - - - ii

Outline - - - - - iii

Introduction - - - - - 1

DNA Sequencing Methods - - - - 3

Uses of DNA Sequencing - - - - 7

Diagnostics - - - - - 8

Molecular Biology - - - - - 9

Forensics - - - - - 10

Conclusion - - - - - 10

Recommendation - - - - - 11

References - - - - - 12

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 INTRODUCTION

DNA sequencing is the determination of precise order of nucleotides [Adenine (A),

Guanine (G), Cytosine (C), Thymine (T)] in a DNA molecule/genome. It includes

many methods that have evolved over a period of time and revolutionized the

biological research, medical diagnostics, forensic sciences and biotechnology. The

focus of this review will be the scientific basis of these methods, how these have

evolved over past few decades, their impact and role in rapid advancement of research

and where the future of sequencing might lead (Watson and Crick, 2000).

Watson and Crick proposed the structure of DNA in their original paper (Watson and

Crick, 2000). It is now known that in this double helix, each strand is composed of a 2'

deoxyribose sugar which is a pentose; its 1' carbon binds one of four nitrogenous

bases while phosphate group at 5' carbon binds hydroxyl group at 3' carbon of next

pentose in sequence. And then its usual binding of adenine with thymine and guanine

with cytosine. Knowledge of this atomic level structure is critical in understanding

various sequencing methods.

Despite discovery of DNA structure, it was only until late 1970s that reliable

techniques were developed to sequence it. Chemical cleavage of DNA for sequencing

was developed by Maxam and Gilbert in late 1970s (Gaastra, 2000, Maxam, and

Gilbert 2001). Owing to complexities of procedure, extensive use of hazardous

chemicals and autoradiography, and short read lengths (less than 500 bp); it is now

obsolete4 and is of historical interest only.

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DNA (Deoxyribonucleic Acid) sequencing is to determine the order of four chemical

building blocks called “bases” that makes up DNA molecule. DNA sequence is a

genetic information that is carried out in specific DNA segment. This DNA sequence

information can be used to determine which stretches of DNA that contain genes and

which transmit supervisory instructions, turning genes on or off and most importantly,

sequence data can highlight variations in a gene that may cause disease. In DNA

sequence, double helix, the four chemical bases constantly bond with the same

paradigm to form base pairs. For instance, adenine (A) always sets with thymine (T);

cytosine (C) always sets with guanine (G). This paring is the basis for the mechanism

by which DNA molecules imitate when cells divide. Human genome contains about 3

billion base pair sets that have instructions for making and maintaining a human being

(Carl, et. al. 2016).

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DNA SEQUENCING METHODS

There are two main types of DNA sequencing. The older, classical chain termination

method is also called the Sanger method. Newer methods that can process a large

number of DNA molecules quickly are collectively called High-Throughput

Sequencing (HTS) techniques or Next-Generation Sequencing (NGS) methods.

Sanger Sequencing

The Sanger method relies on a primer that binds to a denatured DNA molecule and

initiates the synthesis of a single-stranded polynucleotide in the presence of a DNA

polymerase enzyme, using the denatured DNA as a template. In most circumstances,

the enzyme catalyzes the addition of a nucleotide. A covalent bond, therefore, forms

between the 3′ carbon atom of the deoxyribose sugar molecule in one nucleotide and

the 5′ carbon atom of the next. This image below shows how this bond is formed.

(Shiv Kumar, et, al. 2012)

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A sequencing reaction mixture, however, would have a small proportion of modified

nucleotides that cannot form this covalent bond due to the absence of a reactive

hydroxyl group, giving rise to the term ‘dideoxyribonucleotides’, i.e., they do not have

a 2’ or 3’ oxygen atom when compared to the corresponding ribonucleotide. This

would terminate the DNA polymerization reaction prematurely. At the end of multiple

rounds of such polymerizations, a mixture of molecules of varying lengths would be

created.

In the earliest attempts at using the Sanger method, the DNA molecule was first

amplified using a labeled primer and then split into four test tubes, each having only

one type of ddNTP. That is, each reaction mixture would have only one type of

modified nucleotide that could cause chain termination. After the four reactions were

completed, the mixture of DNA molecules created by chain termination would

undergo electrophoresis on a polyacrylamide gel, and get separated according to their

length.

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In the image above, a sequencing reaction with ddATP was electrophoresed through

the first column. Each line represents a DNA molecule of a particular length, the result

of a polymerization reaction that was terminated by the addition of a ddATP

nucleotide. The second, third and fourth columns contained ddTTP, ddGTP, and

ddCTP respectively.

With time, this method was modified so that each ddNTP had a different fluorescent

label. The primer was no longer the source of the radiolabel or fluorescent tag. Also

known as dye-terminator sequencing, this method used four dyes with non-

overlapping emission spectra, one for each ddNTP.

High Throughput Sequencing

High Throughput Sequencing (HTS), also known as Next-Generation Sequencing

(NGS), refers to a set of advanced sequencing technologies that allow for the rapid

and cost-effective sequencing of large amounts of DNA or RNA. Unlike traditional

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Sanger sequencing, which sequences one DNA fragment at a time, HTS can process

millions of fragments simultaneously, enabling comprehensive genomic analysis.

Sanger sequencing continues to be useful for determining the sequences of relatively

long stretches of DNA, especially at low volumes. However, it can become expensive

and laborious when a large number of molecules need to be sequenced quickly.

Ironically, though the traditional dye-terminator method is useful when the DNA

molecule is longer, high-throughput methods have become more widely used,

especially when entire genomes need to be sequenced. (Jacqueline Morris, et. al.

2017)

There are three major changes compared to the Sanger method. The first was the

development of a cell-free system for cloning DNA fragments. Traditionally, the

stretch of DNA that needed to be sequenced was first cloned into a prokaryotic

plasmid and amplified within bacteria before being extracted and purified. High

throughput sequencing or next-generation sequencing technologies no longer relied on

this labor-intensive and time-intensive procedure.

Secondly, these methods created space to run millions of sequencing reactions in

parallel. This was a huge step forward from the initial methods where eight different

reaction mixtures were needed to produce a single reliable nucleotide sequence.

Finally, there is no separation between the elongation and detection steps. The bases

are identified as the sequencing reaction proceeds. While HTS decreased cost and

time, their ‘reads’ were relatively short. That is, in order to assemble an entire

genome, intense computation is necessary (Eugene, et al., 2016).

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The advent of HTS has vastly expanded the applications for genomics. DNA

sequencing has now become an integral part of basic science, translational research,

medical diagnostics, and forensics.

USES OF DNA SEQUENCING

Traditional, chain-termination technology and HTS methods are used for different

applications today. Sanger sequencing is now used mostly for de novo initial

sequencing of a DNA molecule to obtain the primary sequence data for an organism

or gene. The relatively short ‘reads’ coming off an HTS reaction (30-400 base pairs

compared to the nearly a thousand base pair ‘reads’ from Sanger sequencing methods)

make it difficult to create the entire genome of an organism from HTS methods alone.

Occasionally, Sanger sequencing is also needed to validate the results of HTS.

On the other hand, HTS allows the use of DNA sequencing to understand single-

nucleotide polymorphisms – among the most common types of genetic variation

within a population. This becomes important in evolutionary biology as well as in the

detection of mutated genes that can result in disease. For instance, sequence variations

in samples from lung adenocarcinoma allowed the detection of rare mutations

associated with the disease. The chromatin binding sites for specific nuclear proteins

can also be accurately identified using these methods (Carl, et al. 2016)

Overall, DNA sequencing is becoming an integral part of many different applications.

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DIAGNOSTICS

Genome sequencing is particularly useful for identifying the causes of rare genetic

disorders. While more than 7800 diseases are associated with a Mendelian inheritance

pattern, less than 4000 of those diseases have been definitively linked to a specific

gene or mutation. Early analysis of the exon-genome, or exome, consisting of all the

expressed genes of an organism, showed promise in identifying the causal alleles for

many inherited illnesses. In one particular case, sequencing the genome of a child

suffering from a severe form of inflammatory bowel disease connected the illness to a

mutation in a gene associated with inflammation – XIAP. While the patient initially

showed multiple symptoms suggestive of an immune deficiency, a bone marrow

transplant was recommended based on the results of DNA sequencing. The child

subsequently recovered from the ailment (Drmana, et al. 2010).

In addition, HTS has been an important player in developing a greater understanding

of tumors and cancers. Understanding the genetic basis of a tumor or cancer enables

doctors to have an extra tool in their kit for making diagnostic decisions. The Cancer

Genome Atlas and International Cancer Genome Consortium have sequenced a large

number of tumors and demonstrated that these growths can vary vastly in terms of

their mutational landscape. This has also given a better understanding of the kind of

treatment options that are ideal for each patient. For instance, the sequencing of the

breast cancer genome identified two genes – BRCA1 and BRCA2 – whose pathogenic

variants have an enormous impact on the likelihood of developing breast cancer.

People with some pathogenic alleles even choose to have preventive surgeries such as

double mastectomies (Drmana, et al. 2010).

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MOLECULAR BIOLOGY

DNA sequencing is now an integral part of most biological laboratories. It is used to

verify the results of cloning exercises to understand the effect of particular genes. HTS

technologies are used to study variations in the genetic compositions of plasmids,

bacteria, yeast, nematodes or even mammals used in laboratory experiments. For

instance, a cell line derived from breast cancer tissue, called HeLa, is used in many

laboratories around the world and was earlier considered as a reliable cell line

representing human breast tissue. Recent sequencing results have demonstrated large

variations in the genome of HeLa cells from different sources, thereby reducing their

utility in cell and molecular biology.

DNA sequencing gives insight into the regulatory elements within the genome of

every cell, and the variations in their activity in different cell types and individuals.

For instance, a particular gene may be permanently turned off in some tissues, while

being constitutively expressed in others. Similarly, those with susceptibility for a

specific ailment may regulate a gene differently from those who are immune. These

differences in the regulatory regions of DNA can be demonstrated through sequencing

and can give insight into the basis for a phenotype (Eugene, et al., 2016).

Recent advances have even allowed individual laboratories to study structural

variations in the human genome – an undertaking that needed global collaboration two

decades ago.

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FORENSICS

The ability to use low concentrations of DNA to obtain reliable sequencing reads has

been extremely useful to the forensic scientist. In particular, the potential to sequence

every DNA within a sample is attractive, especially since a crime scene often contains

genetic material from multiple people. HTS is slowly being adopted in many forensics

labs for human identification. In addition, recent advances allow forensic scientists to

sequence the exome of a person after death, especially to determine the cause of death.

For instance, death due to poisoning will show changes to the exome in affected

organs. On the other hand, DNA sequencing can also determine that the deceased had

a preexisting genetic ailment or predisposition. The challenges in this field include the

development of extremely reliable analysis software, especially since the results of

HTS cannot be manually examined (Ley, et al. 2018).

CONCLUSION

DNA sequencing has fundamentally transformed our understanding of genetics,

biology, and medicine. By enabling the precise determination of nucleotide sequences,

it has opened new avenues for research and innovation across multiple fields. The

implications of this technology are vast and multifaceted, ranging from personalized

medicine to significant advancements in agricultural biotechnology, evolutionary

studies, and forensic science.

RECOMMENDATION

DNA sequencing is a groundbreaking technology that enables the analysis of the nucleotide

sequence in DNA, providing significant insights into genetics, medicine, and biology. It is

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advisable to utilize next-generation sequencing (NGS) due to its high efficiency, rapid

processing, and cost advantages over traditional techniques. NGS can support extensive

genomic research, including whole-genome, targeted, and RNA sequencing, helping to

identify genetic variations linked to diseases, inform personalized medicine, and discover

potential therapeutic targets. Beyond its scientific applications, DNA sequencing raises

ethical issues related to privacy, consent, and the risk of genetic discrimination. As these

technologies evolve, it is essential to develop strong regulatory frameworks and guidelines to

address these challenges while encouraging the responsible application of genetic data.

Additionally, collaboration among researchers, healthcare providers, and policymakers is

crucial for maximizing the benefits of DNA sequencing for public health, ensuring equitable

access while protecting individual rights.

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 References

Carl W Fuller, Shiv Kumar, Mintu Porel, et al. (2016) Real-time single-molecule electronic
DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.
Proc Natl Acad Sci U S A 2016;113:5233-8.

Drmanac R, Sparks AB, Callow MJ, Halpern AL, Burns NL, Kermani BG, et al. (2010)
Human genome sequencing using unchained base reads on self-assembling DNA
nanoarrays. Science 2010; 327:78-81.

Eugene Paulechka, Tsjerk A Wassenaar, Kenneth Kroenlein, Andrei Kazakov, Alex

Smolyanitsky.(2016) Nucleobase-functionalized graphene nanoribbons for accurate
high-speed DNA sequencing. Nanoscale 2016; 8:1861-7.

Gaastra W. (2000) Chemical cleavage (Maxam and Gilbert) method for DNA sequence
determination. Methods Mol Biol 00; 2:333-41.

Jacqueline Morris, Young-Ji Na, Hua Zhu, et al. (2017) Pervasive within-Mitochondrion
Single- Nucleotide Variant Heteroplasmy as Revealed by Single-Mitochondrion
Sequencing. Cell Rep 2017;21:2706-13.

Ley TJ, Mardis ER, Ding L, Fulton B, McLellan MD, Chen K, et al. (2018) DNA sequencing
of a cytogenetically normal acute myelogenous leukemia genome. Nature 2018;
456:66-72.

Maxam AM, Gilbert W. A (2001) new method for sequencing DNA. Proc Natl Acad Sci
USA 01; 74:560-4.

Shiv Kumar, Chuanjuan Tao, Minchen Chien, et al. (2012) PEG-Labeled Nucleotides and
Nanopore Detection for Single Molecule DNA Sequencing by Synthesis. Scientific
Reports 2012;2.

Watson JD, Crick FH. (2000) Molecular structures of nucleic acids. Nature 00; 71:737.

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