2. What is DNA Sequencing?
We understand DNA fragments as a nucleic acid
bases with sugar phosphate molecules attached to
it. When these fragments are put linked together,
they create the double helix that Watson & Crick
announced in 1953. We will be examining how
technological advances have allowed scientists to
order and catalogue the information as fragments, in
a process called DNA sequencing.
Currently, sequencing is carried out by machines
and this presentation will focus on automated
processes of DNA sequencing.
http://www2.hkedcity.net/sch_files/a/abc/abc-lnk/public_html/Biotech/dna_molecule1.html
3. The DNA Sequencing Machine: Early Stage
How is DNA sequencing possible? Through the
double helix model, we can understand that the
complementing pair structure allows for one
strand to inform what the complete double
strand would look like. This concept is what
allowed for the first automated technique --
dideoxyribonucleotide chain termination
sequencing.
One strand of DNA fragment is set as a
template for synthesis of a nested set of
complementary fragments. These are further
analyzed to yield the sequence.
http://bio1151.nicerweb.com/Locked/media/ch20/termination.html
4. The DNA Sequencing Machine: Next Generation
After dideoxy chain termination, sequencing by
synthesis was adopted because it allowed to
order more nucleotides in a significantly shorter
period of time. The older method could order
12,000 base pairs in 10 hours; the new one
could order up to 900 million base pairs in 10
hours.
http://joe.endocrinology-journals.org/content/201/1/1/F2.large.jpg
5. The DNA Sequencing Machine: Next Generation
Sequence by synthesis works through
amplifying DNA fragments that have been
processed through an aqueous solution. It is
copied so that the surface of a single bead of
the solution can have 106
copies of the
fragment. These copies are all 5’ ends and the
beads are placed in wells to be matched with
their 3’ ends. A solution containing one of the
four nucleotides is washed over the entire plate.
Each nucleotide is coded with a different light-
emitting flash that indicates when it has been
added to a match in the well. The fragments are
then organized in a “flow-gram”.
http://www.mayomedicallaboratories.com/articles/communique/2010/05.html
6. The DNA Sequencing Machine: Third Generation
A single, very long DNA molecule is sequenced
on its own through a nanopore. This is a very
small pore, like a protein channel pore,
embedded in a lipid membrane. Scientists are
detecting the bases individually by the
observing the effect on ions and flow of an
electrical current that is being charged through
the pore. Other examples of third generation
sequencing include artificial membranes and
nanopores.
http://www.engadget.com/2010/12/24/nanopore-dna-sequencing-technique-promises-entire-
genome-in-minu/
7. What is Amplification?
Amplifying simply means to make copies of.
For example, when examining a particular trait,
an investigator may amplify a targeted segment
of the DNA in order to isolate which nucleotides
are at causing the gene expression. Without
amplification, copies of a cloned gene cannot be
used in basic research or to endow another
organism with a new ability. Since one gene is
only a very small part of the total strand, the
availability to amplify DNA fragments is crucial
for any application involving a single gene.
http://www.degruyter.com/view/j/bmc.2013.4.issue-6/bmc-2013-
0026/graphic/bmc-2013-0026_fig1.jpg
8. DNA Cloning
To study specific genes, scientists have
developed methods for preparing DNA
segments by cloning them.
They use bacteria cells that contain plasmids.
They take DNA segments and insert that
segment into bacterial plasmid, which has been
modified for efficient cloning. This combination
of foreign DNA and bacterial plasmid is called a
recombinant DNA molecule.
The plasmid is returned to a bacterial cell where
it reproduces a clone of cells.
http://fhs-bio-wiki.pbworks.com/f/1265935367/8363.nfg021.jpg
9. Why Plasmids?
The plasmid is an ideal cloning vector. A
cloning vector indicates a DNA molecule that
can carry foreign DNA into a host cell and
replicate endlessly.
Readily obtained through commercial supply,
plasmids from E coli cells are commonly
manipulated to form recombinant plasmids. This
is done in vitro. The host cells grow in a
prepared culture and contains many copies of
the gene of interest. A protein can then be
extracted from thee genetic copies and
eventually altered to make medicine or to alter
gene expression to solve problems.
http://www.mun.ca/biology/desmid/brian/BIOL4900/1828B.JPG
10. Polymerase Chain Reaction
When making copies of gene sequences, we
use a technique called a polymerase chain
reaction. This is a three-step cycle that
produces DNA copies on an exponential scale.
Step 1: Temperature is manipulated to separate
the strands and allow primers and DNA
polymerase to make two pairs of complete
strands.
Step 2 and 3: The process is repeated so that
you have made 2 copies, then 4, then 8, etc.
http://d3qpq7e7yxjovl.cloudfront.net/content/ajpadvan/28/2/44/F1.large.jpg
11. Nucleic Acid Hybridization
Hybridization is key to establishing and
clarifying segments in DNA. Without this
process, copies could not be created. The
hybridization process takes the base pairing
of one strand and combines it to the
complementary sequence on a strand from
another nucleic acid molecule. This can
happen in an aqueous solution, in a gel, or even
on special paper (nitrocellulose paper).
We use in situ hybridization when fluorescent
dyes are applied to specific chromosomal
segments that are being investigated.
http://www.intechopen.com/source/html/38366/media/image2.jpeg
12. Restriction Enzymes
Restriction enzymes are important
components of the bacterial cell that protect it
from the control sequences of foreign DNA that
can launch disruptive protocols. There are many
restriction enzymes and each has specific
sequences of their own that correspond with a
restriction site. The sites represent areas
where DNA strands are cut off and can be
combined with other DNA fragments, like within
plasmids. Restriction enzymes are coded with 4
to 8 nucleotide pairs and we call these
sequences between restriction enzymes
restriction fragments.
https://pmgbiology.files.wordpress.com/2014/11/image001.gif
13. Sticky Ends and DNA Ligase
When restriction enzymes cleave the sugar-
phosphate backbones in the two DNA strands,
the resulting double-stranded restriction
fragments have what is referred to as a “sticky
end”. The sticky ends are where DNA fragments
from another source, such as a bacterial
plasmid, can be paired with the foreign DNA.
The bonds formed are temporary until they are
sealed by the enzyme DNA ligase. Ligase
catalyzes the formation of covalent bonds that
bind the sugar-phosphate backbones of DNA
strands that were previously broken by the
restriction enzymes.
http://swh.schoolworkhelper.netdna-cdn.com/wp-content/uploads/2010/07/ch5f1.jpg?e6ecea
14. Cloning Eukaryotic Genes
Eukaryotic genes can be cloned despite their
differences from bacterial host cells because the
controls meant to order sequences get
bypassed. An expression vector is used above
the restriction site so that the host will identify
the promoter and begin copying, even if the
information is from an eukaryotic gene.
Eukaryotic cells also carry non-coding segments
that bacterial cells do not. This is bypassed by
using only complementary DNA by copying from
mRNA which only contains exons.
http://classes.midlandstech.
edu/carterp/Courses/bio225/chap09/10th_ed_figures/figure_09_09_labeled.jpg
15. Electroporation
Another method of introducing foreign
eukaryotic DNA into a bacterial cell is through a
process called electroporation. This is when a
short electrical pulse runs through an aqueous
solution that contains both cells. The pulse
punctures small holes in the plasma membrane
of the bacterial cells. Through these channels,
the foreign DNA can enter.
http://www.bioelectrochemical-soc.org/img/electroporation-1.jpg
16. Gel Electrophoresis
It is useful, after DNA copies have been made,
to separate the bacterial plasmid from the
foreign DNA. Thus, the DNA can be examined
as a fragment. To study fragments of various
lengths, researchers can use a process called
gel electrophoresis. The gel is a polymer used
as a molecular sieve to separate out a range of
nucleic acids, based on their length by running
an electrical charge through the gel. The
shortest fragments will travel the fastest and
farthest from one charged end to the other
because they are lighter.
http://bio1151.nicerweb.com/Locked/media/ch20/20_08GelElectrophoresis.jpg
17. Gel Electrophoresis
The agarose gel is submerged in saltwater that
will conduct electricity. Slots are pre-formed in
the gel and pipettes will inject the DNA into
those wells. Researchers will add dye so that
they can visualize the molecules as they run
through the gel. DNA has a general negative
change and this will make them propel to the
positive charge of the other side of the gel.
When UV light is shone onto the gel, the dye
gives off a fluorescence that is captured on film.
DNA fragments are compared to known
fragment sizes.
https://upload.wikimedia.org/wikipedia/commons/e/e6/DNAgel4wiki.png
18. Evolutionary Ancestry
What can we conclude from the bacterial cell’s
ability to produce proteins encoded in eukaryotic
cells? We can look backwards at our course
timeline to when we answered a similar question
about how these two type of cells originated. We
can theorize that because of their shared
mechanism of gene expression, they had a
common ancestor, through which, we can still
witness evolutionary roots. This also means that
given the right conditions, genes that are taken
from one species can function and be expressed
in an entirely different species.
http://s1251.photobucket.com/user/celine046/media/piggies_zps2bceb3b5.jpg.html
19. The Human Genome Project
The Human Genome Project is a government-
led initiative to map the entire sequence of the
human genome. The main and most immediate
benefits of mapping the human genome was the
information available to medical professionals
who would be able to interpret the data.
Research has been on the rise about diseases
that are inherited genetically. If there is an
identifiable segment of DNA that links with a
disease, then there may be a way to prevent
these ailments from occurring. This project has
led to offshoots such as the Cancer Genome
Project, which will be pivotal to cancer-related
trials, drugs, and prevention. https://www.achieversdaily.com/wp-content/uploads/2015/04/sequencing_explosion.jpg
20. Genetic Engineering
This presentation brings all of the tools of DNA
sequencing into further detail and concludes
with a question outside the realm of pure
scientific inquiry: the issue of direct
manipulation of genes for practical purposes.
Regardless of how we as a species use genetic
engineering, no one can deny the
groundbreaking medicinal achievements that
have been made due to our ability to sequence
and order DNA. Ultimately, our descendants will
decide how we use future technology but for
now, we can only hope that we will obtain
progress in how we battle genetic diseases.
https://s3.amazonaws.com/lowres.cartoonstock.com/science-genetic_engineering-
genes-perfect-perfect_men-scientists-mban1450_low.jpg
