DNA Sequencing Principles and Aplication [610013]

DNA Sequencing Principles and Aplication
Course: Princibles And Technicues Of Working In Genomics
Year/Semester: 2017-18/ Spring
Student: [anonimizat]

1.İntroduction
Completion of the human genome project is the beginning of the
postgenomic field and the system biology approach has gained great
importance. Regulatory networks associated with cellular hemostasis,
development and disease progression in the human genome and functional
elements playing a role in these networks have begun to be identified. The
identification of these functional elements in the genome that regulates
cellular activities and their variation among populations is also the basis
for the development of person-specific treatment.
In parallel with the development of recombinant DNA techniques, which
allow a large amount of pure DNA to be obtained from any organism,
DNA sequencing methods have also been developed. Research on DNA
sequence analysis, which began in the 1960s, has mainly developed in the
following way (Zülal, 2001).
History
In 1965, Robert HOLLEY performed sequence analysis of a 74 nucleotide
tRNA molecule. In 1977, two different DNA sequence analysis methods
were found by Allan MAXAM – Walter Gilbert and Frederick SANGER.
In 1982 Akiyoshi proposed WADA DNA sequence analysis to be
automated and robots started to be developed. In 1986, from California
Institute of Technology (Caltech) Leroy HOOD and Llyod SMITH found a
fully automatic machine to be used in DNA sequence analysis. In 1990,
Edward UBERBACHER began using GRAY, a gene finding program. In
1992 DNA sequence analysis of chromosome 21 was completed.In 1995,
the first DNA sequence of Haemophilus influenzae was published by
Craig VENTER, Claire FRASER and Hamilton SMITH. In 1996, a
national consortium published a DNA sequence of S.cerevisiae, a yeast
strain. In 1998, the DNA sequence of Caenorhabditis elegans was
described by scientists at the Sanger Center and Washington University. In
1999, the DNA sequence of human chromosome 22's was completed by
scientists from the United Kingdom, Japan and the United States. In 2000
it was cooperated with Celera and the DNA sequence of Drosophila
melanogaster was described by the universities. In June 2000, the Human
Genome Project participants and Celera announced that they completed
the draft of the human gene map. Arabidopsis thaliana has been the first
plant in the DNA sequence described in 2000.David PAGE and his
colleagues at the Whitehead Institute in 2003 completed sequence analysis

of the Y chromosome.
DNA sequence analysis has 2 methods that are still used today. These;
1. Maxam and Gilbert's chemical fracture method
2. Sanger-Coulson's chain ending method
Bu iki yöntemden Sanger – Coulson'un yöntemi günümüzde daha yaygın
olarak kullanılmaktadır.
1. Maxam and Gilbert's chemical fracture method
The principle of the method developed by Allan MAXAM and
Walter Gilbert is that hydrazine, dimethyl sulphate or formic acid
specifically changes the bases in the DNA and then breaks the chain at the
point where the added piperidinein nucleotides are added (Sambrook, et
al., 1989 ). In this method, the DNA to be detected by the nucleotide
sequence is first labeled with 5'-end 32P or with a fluorescent dye. The two
strands of DNA are separated from each other or the DNA is cleaved with
a suitable restriction enzyme so that only one side of the DNA is marked.
In the second step, the DNA molecules are separated into four test tubes
and the reactions necessary to modify and break the A, C, G or T
nucleotides are performed. By giving a limited time for the reaction, DNA
fragments broken out from target nucleotides at different positions in each
tube are obtained. As a result, a series of DNA fragments with the same 5'-
positions but different lengths are obtained according to the position at
which they are broken. Obtained DNA fragments are separated by size by
gel electrophoresis, and autoradiography is applied to display the bands
(Klug et al., 2000).
2. Sanger-Coulson's chain ending method
Another method used in DNA sequencing is the chain termination
method developed by Fred SANGER et al. (Sanger et al., 1977). This
method is based on enzymatic DNA synthesis and is the most widely used
DNA sequencing technique of our today. In this method, the DNA strand
to be detected in the sequence is used as a template for the newly
synthesized yarn. One of Klenov, Taq DNA polymerase, reverse
transcriptase or secuenase enzymes can be used to provide DNA synthesis.
The method is based on the ability of the DNA polymerase to use dNTPs
(deoxyribonucleoside triphosphate ) as well as ddNTPs
(dideoxyribonucleoside triphosphate) that do not carry an OH group at the

3 'position, as well as dNTPs. Incorporation of a ddNTP into the
synthesized DNA stops the synthesis because there is no OH group at the
3' position. When sequence analysis is performed, four separate reaction
mixtures are prepared. Each mixture contains a template DNA chain, a
primer, four of the dNTPs and a small amount of ddNTPs. For each
specific chain termination, a different ddNTP is found in each reaction.
Since very small amounts of modified nucleotides are used in each
reaction, the new chain synthesis randomly terminates, resulting in a series
of DNA fragments (Klug et al., 2000).
a) dATP b) ddATP molecules
In reactions are run side by side on the gel electrophoresis on the resulting
DNA fragments. With the effect of the applied electric field, the DNA
particles form a ladder image on the gel with the shortest foremost. On the
gel according to the marking method, the detected particles are read
according to the type of ddNTP put into the reaction mixture (Klug et al.,
2000).
These two techniques described above are appropriate and sufficient in
accordance with the requirements of the present period and are still in use
today, but nowadays, new generation DNA sequencing analysis techniques
have been developed since these two methods can not be used for both
analysis of accuracy and analysis of long DNA molecules and most
importantly time consuming technique. This new generation of the
sequencing techniques is a technique that is fast, easy and has a very high
accuracy.

New Generation Sequencing Techniques
1. Pyrosequencing Method
In 1996, Ronaghi and his colleagues developed the "Pyrosequencing"
method based on the principle of "sequencing by synthesis" (Ronaghi et
al., 1999). Pyrosequencing has taken the place of traditional Sanger
sequencing thanks to its high transaction volume and low cost.
Pyrosequencing is based on the detection of DNA polymerase activity
based on a chemiluminescent enzyme. The sequancing reaction is the
synthesis of the complementary strand (cDNA) on the single-stranded
(ssDNA) DNA to be sequenced. First, the nebulization of double-stranded
DNA (separation into small fragments of 400-800 bp) is performed.
Adapters carrying sequencing primers are added to these fragments.
Emulsion-based clonal amplification (em-PCR) of DNA fragments
carrying adapters is performed in microreactors. As a result of this step,
approximately 10 million copies of the extracted DNA fragments are
sequenced in a plate containing approximately 3.6 million wells by
sequencing. In the case that A, C, G, T nucleotides complementary to the
template DNA sequence are flowing in succession on a single-stranded
template DNA that is fixed with the sequencing primer, a light is generated
in the medium. It is determined which nucleotide linkage of the
chemiluminescent signal that produces this light occurs. Immobilized
ssDNA is incubated with DNA polymerase, ATP sulphyrylase, luciferase,
apirase, APS and luciferin. The DNA polymerase allows the
pyrophosphate (PPi) to become clear when complementary nucleotides are
present during the nucleotide flow. The ATP sulphyrylase enzyme
quantitatively converts this PPi to ATP. ATP allows the conversion of
luciferin to oxylus ferment through the luciferase enzyme. Oxyluciferin
creates a visible light. The intensity of this light is directly proportional to
the amount of ATP, in which only single nucleotide repeats
(homopolymer) on the same sequence are detected. The resulting light is
recorded by the CCD camera and converted into sequence data (florogram)
using the computer program (Ronaghi M., 2001).

2. Sequencing with cyclic reversible termination
DNA molecules are first amplified (bridge amplification) by
attaching to the primers on the slide. Four types of dNTPs are added and
the ddNTPs that do not enter the structure of the newly synthesized thread
are removed with washed. Unlike pyrosequencing, DNA, after each wash,
is a nucleotide increases. After fluorescently labeled nucleotides are
detected by the camera, the final 3 'end is chemically removed and the next
cycle is passed (Mardis ER., 2008).
3. Sequencing by ligation
In this method, emulsion PCR is carried out as in pyrosequencing.
The primers hybridize to the template yarn with the help of adapter arrays
as they are in the pyrosequencing. In this method, the DNA ligase enzyme
is used instead of the DNA polymerase. Fluorescently labeled
oligonucleotide probes are used, the probes hybridize to the template DNA
and enter the primer ligation. After fluorescence detection, the signals of
the oligonucleotide probes are switched to a new ligation cycle by cutting
the 5 'phosphate backbone. The DNA sequence is determined by repeating
the ligation, detection and cleavage cycles (Schuster SC., 2008)
4. Ion semiconductors sequencing
This method is based on the detection of hydrogen ions which occur
during the polymerization of DNA. Single nucleotide flow to the micro
well containing a single template DNA sequence is applied. If the given
nucleotide is complementary to the template, it is absorbed into the
structure of the newly formed thread and the hydrogen ion is released.
Hydrogen ions are detected by hypersensitive ion sensors. In the presence
of homopolymer repeats in the template DNA, the single strand will enter
the structure of more than one nucleotide new strand and the DNA
sequence is determined by obtaining more electronic signals than the
number of released hydrogen ions (Rushk N., 2011).

5. Nano sequencing
In this method, it is based on the sequencing by synthesis. As a direct
molecular sensor, a different bio-engineered polymerase and dNTPs are
used. The synthesis of a single DNA molecule at each nanomachine can be
monitored in real time (Treffer at al. 2010).
Result
DNA sequencing methods are methods used to determine DNA primer
sequences and nucleotide base sequencing. DNA sequencing methods
have used a lot of information about gene structure and genetic control
mechanisms. Thanks to these methods, gene maps of many organisms
have been extracted and continued to be extracted. It is usually used in the
determination of gene mutations or recombinant DNA formations.
Even in today's conditions, DNA samples taken from in vitro fertilized
embryos in couples carrying hereditary diseases are analyzed by these
methods and carrier embryos are sifted and non-carrier embryos are born.
The necessity of new generation DNA sequencing methods with the reason
that Maxam and Gilbert's chemical breakage methods and Sanger-
Coulson's chain termination method, which are the first of DNA
sequencing methods, are inadequate in today's conditions and have many
disadvantages.
The disadvantages are as follows;
– The complexity of methods
– Not to be used with biological kits (Only Maxam and Gilbert's chemical
breakage methods)
– Use of hazardous chemical substances
– Both methods are long and time consuming
– They are unfavorable for large genome sequencing
Due to such reasons, new generation DNA sequencing methods have been
developed and continue to be developed and the usage areas are increasing
day by day.

References
1. Zülal, A., 2001, İnsan Genomu, kalıtım șifresinin peșinde 136 yıl,
Tübitak Yayınları, Mart; 5-11
2. Sambrook, J. Fritsch, E.F., Maniatis, T. 1989 Molecular Cloning, a
laboratory manual. Cold spring harbor laboratory Press New York.
3. Klug, S.W., Cummings, W.R., 2000 Concept of Genetics, Prentice Hall,
New Jersey 745 p..
4. Sanger, f., Nicklen, S., Coulson, A.R., DNA sequencing with chain-
terminating inhibitors. 1977, Proceedings of the National Academy of
Sciences, 74, 5463-7.
5. Ronaghi M, Nygren M, Lundeberg J, Nyren P. Analyses of secondary
structures in DNA by pyrosequencing. Anal Biochem. 1999; Feb
1;267(1):65-71
6. Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome
Res. 2001; Jan;ll(l):3-ll
7. Mardis ER. Next-generation DNA sequencing methods. Annu Rev
Genomics Hum Genet 2008; 9: 387^402
8. Schuster SC. Next-generation sequencing transforms today's biology.
Nat. Methods 2008. 5 (1): 16-8
9. Rusk N. Torrents of sequence. Nature Methods 2011; 8(1)44
10. Treffer R, Deckert V. Recent advances in singlemolecule sequencing.
Current Opinion in Biotechnology 2010; 21( 1): 4-11