DNA sequencing technology

DNA Sequencing
UE Gestion de l’innovation
Teacher: Christophe Lécuyer
Anya Rahmoune
L3 Sciences de la Vie-Gestion
2019-2020

SUMMARY
Introduction
II – History of DNA sequencing
III – Pharmacogenomics
IV – Direct-to-consumer genetic testing: case study of 23andme
IV – Genetic sequencing, ethics and society
Conclusion
Bibliography
Appendixes

Introduction
Since its discovery, DNA has fascinated scientist and general public alike. Indeed, a large part of a person’s
information can be found in its DNA: their biological sex, their ancestry, their physical appearance, sometimes
even their medical condition. Genetic makeup is part and parcel of who we are. It’s why DNA sequencing was
crucial to the pursuit of research in Life Sciences. Some scientists go as far as to say DNA sequencing
technology is such an important innovation, it is akin to that of the microscope. Subsequently, this technology
was embraced by many industries in the Life Sciences field: medicine, virology, evolutionary biology, forensics,
etc. and even spawned new ones, two of which we will study in this essay: pharmacogenomics and direct-to-
consumer genetic testing.
The question is, how did DNA sequencing technology came to be and how did it become essential and
ubiquitous in the Life Sciences industry, adopted as it is by every laboratory and company in this field? To
understand how this came to be, we will start with a retelling of the discovery of genetic testing technology.

I – History of DNA sequencing
DNA sequencing as an innovation was very much incremental. Several milestones had to be reached to get
to the technology we have today. Listing each one would be too long, and it is not the point of this essay, so
we’ll only name the most important ones.
An important figure in DNA sequencing is Frederick Sanger, a British biochemist who received two Nobel
Prizes: the first for “his work on the structure of proteins, especially that of insulin”, the second, he shared
with Walter Gilbert for “their contributions concerning the determination of base sequences in nucleic acids”
– that is to say, DNA sequencing. Sanger did not discover DNA sequencing methods immediately, he first had
to sequence a protein, insulin, then RNA, and finally DNA.
Sequencing of Insulin
Frederick Sanger obtained his Ph.D in 1943 in Cambridge, working on the metabolism of the amino acid
lysine in the animal body. The same year, he got his first job, a position in the Biochemistry department of
Cambridge offered by A.C. Chibnall. Chibnall was interested in insulin, of which he determined, with
colleagues, the amino acid composition of its bovine form. The structure of insulin, that is to say, the exact
order of these amino acids, was however still a mystery to biologists.
Chibnall put Sanger up to the task of investigating the end groups of the
protein, which would later lead to research on its structure. Sanger tried several
techniques and molecules before he managed, thanks to his connections, to get
his hands on dinitrofluorobenzene (DNFB), synthesised at the time for the war
effort. It worked, and he published his first paper. It was not the discovery of the
entire sequence yet, but it was a start.
Sanger later found insulin was made of two chains, that he called chain A et
chain B. With Hans Tuppy and later Ted Thompson, Austrian and Australian
biochemists, he gradually pieced together the complete structure of insulin from
overlapping sequences.
The only thing missing was the emplacement of the disulfide bonds
1
that
joined the two chains. Using the new technique of paper electrophoresis
2
, Sanger located the disulfide
bridges, thus finally completing the structure of insulin.
Sanger therefore proved proteins are well-ordered molecules and that, by analogy, genes must be, too. For
his work, Sanger received his first Nobel Prize in Chemistry in 1958.
Sequencing of RNA
At the time, nucleic acids, that is to say, DNA and RNA, were still a mystery to scientists. Sanger only got
interested in RNA because DNA remained too difficult to sequence yet. We know now DNA and RNA are made
of pieces called nucleotides, of which there are five types: A (adenine), C (cytosine), G (guanine), T (thymine)
and U (Uracil). We know DNA is exclusively composed of A, C, G, and T, and mRNA
3
can be a perfect mirror to
DNA, with the only difference being every T is replaced by a U in the sequence. Today, we also know, for every
gene of the human genome, the exact order of these nucleotides.
1
Disulfide bonds : a disulfide refers to a functional group with the structure R−S−S−R′. The linkage is also called an SS-bond or
sometimes a disulfide bridge. Source : Wikipedia.
2
Paper electrophoresis : technique used to separate small charged molecules such as amino acids and small proteins.
3
mRNA : single-stranded RNA molecule that corresponds to the genetic sequence of a gene and is read by the ribosome in the
process of producing a protein. Source : Wikipedia.

However, at the time, DNA molecules were too big and complicated for scientists to sequence, with no
known technique to identify the structure. With RNA, on the other hand, it was more manageable. Even if the
simplest RNA viruses contained a few thousand nucleotides, still too big to sequence, tRNAs
4
, a different and
newly-discovered type of RNAs, contained fewer than a hundred.
The appeal was obvious. Sanger’s goal was to not only work out sequence methods for RNA and be the first
to sequence one, but also to reveal the genetic code. Indeed, since mRNA reflects the DNA structure,
sequencing a mRNA corresponding to a protein whose sequence is known would reveal the genetic code.
However, Robert Holley from Cornell University published first his sequence analysis of the 77
ribonucleotides of alanine tRNA from a species of yeast in 1965 and so beat him to it. By 1967, Sanger's group
had determined the nucleotide sequence of a small RNA of 120 nucleotides from bacteria E. coli.
Sequencing of DNA
DNA sequencing was Sanger’s ultimate goal, the consecration of his career. The problem, at the time, was
the unavailability of small-sized DNA or ways to break long DNA into smaller, more manageable pieces.
Sanger chose to work on bacteriophage ØX174 DNA because of its convenience: the phage was single-
stranded and his colleague John Sedat knew how to grow it. He wanted to use DNA polymerase
5
to develop
his DNA sequencing method, but DNA polymerase requires a primer
6
, and such a primer would have to be
synthesized. Thanks to Hans Kössel, a German molecular biologist who worked alongside Khorana, an Indian-
American biochemist who pioneered DNA synthesis methods at MIT, Sanger had access to octanucleotide
primers. So he used an octanucleotide primer and E. coli DNA polymerase.
In the early 1970s, Sanger hired a new technician, Alan Coulson, 20 year old. Because of their shared
reserved nature, a sharp contrast to the outgoing personality of other members of Sanger’s team, Coulson
and Sanger get on marvellously and worked well together.
Sanger and Coulson went on to invent what they called “the plus and minus method” of DNA sequencing.
By 1977, they had published the whole 5400 base pair sequence of bacteriophage ØX174. They also
discovered the number of proteins produced by ØX174 to be much higher than the number of its genes –
which meant that the one gene-one enzyme hypothesis of Beadle and Tatum was false. Gene sequences
overlapped, they just happened to start at different points in the DNA sequence.
This method outperformed other techniques, but it was still too laborious. Inspired by Fred Arthur
Kornberg’s work posing dideoxythymidine triphosphate (ddTTP) as a substrate for DNA polymerase, Sanger
developed another method, the dideoxy chain-termination method, or what is known today as the Sanger
method. In parallel, Maxam and Gilbert were working on another method, perfected around the same time
Sanger invented his. However, the Sanger method used fewer toxic chemicals, lower amounts of radioactivity
and was easier and reliable, so it became the favoured method from the 1980s to the 2000s
7
.
In 1980, Sanger received his second Nobel Prize in Chemistry, which he shared with Gilbert, for “their
contributions concerning the determination of base sequences in nucleic acids”.
Sanger’s method is only the first generation of DNA sequencing method. After that come the second and
third generations, quicker, more precise and more reliable, each time needing less genetic material to work
with.
4
tRNA : necessary component of translation from mRNA to proteins.
5
DNA polymerase : enzymatic complex intervening in DNA replication during cell multiplication.
6
Primer : short single-stranded nucleic acid utilized by all living organisms in the initiation of DNA synthesis. Source : Wikipedia
7
Source : DNA sequencing, Wikipedia.