DNA SEQUENCING

BSMT 2 | S.Y. 2021-2022 Second Semester – FINALS

1970 Discovery of type II restriction enzymes by
Hamilton Smith
List of Contents:
• History of DNA Sequencing 1977 Maxam-Gilbert sequencing
• Introduction Frederick Sanger sequencing
• What exactly is DNA Sequencing?
• DNA Structure 1983 Polymerase chain reaction (PCR), developed by
• In the Beginning Kary B. Mullis is revolutionary technique that
• How is DNA Sequencing performed? enables scientists to rapidly amplify DNA
• Different methods for DNA Sequencing
• Sanger Sequencing
INTRODUCTION
• Max-Gilbert Sequencing
• Application • The term DNA sequencing refers to sequencing
• Advantages methods for determining the order of the nucleotide bases
• Disadvantages - adenine, guanine, cytosine, and thymine - in a molecule
• Conclusion of DNA.
CYTOGENETICS LESSON #9 • Knowledge of DNA sequences has become
DNA SEQUENCING indispensable for basic biological research, other
Lecturer: Mr, Stefano Paolo Jesalva research branches utilizing DNA sequencing, and in
numerous applied fields such as:
HISTORY OF DNA SEQUENCING o Diagnostic
o Biotechnology
o Forensic Biology
o Biological Systematics
WHAT EXACTLY IS DNA SEQUENCING?
• The very basic unit of the human genome is a single DNA
nucleotide. This nucleotide is extremely small and is
made up of miniscule atoms, which creates a challenge
for even an advanced microscope to be used for
detection
• Researchers still, however, need to be able to determine
• The sequencing of DNA molecules began in the 1970s
the sequence of bases in DNA that make up the human
with development of the Maxam-Gilbert method, and
genome. As such, DNA sequencing has been developed
later the Sanger Method
but the process itself is a seemingly complex one.
• (Sanger Method) Originally developed by Frederick
• DNA sequencing involves the determination of the order
Sanger in 1975, most DNA sequencing that occurs in
of DNA bases.
medical and research laboratories today is performed
using sequencers employing variations of the Sanger DNA STRUCTURE
method • In a strand of DNA, there are some simple units known
as nucleotides. These nucleotides have a ‘backbone’ that
consists of sugars and a phosphate group (sugar-
phosphate backbone). The DNA bases can be one of the
four kinds and they are attached to these sugars. These
bases hold important and unique genetic information for
the body. These bases are:
o Adenine (A)
o Thymine (T)
o Cytosine (C)
o Guanine (G)

1953 Discovery of DNA double helix by James

Watson and Francis Crick

1965 Escherichia coli alanine tRNA was the first

nucleic acid molecule to be sequenced

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IN THE BEGINNING
• The very first methods used for DNA sequencing were
created in the 1970s
• During this decade, researchers were only able to
sequence a small number of base pairs
• By 1990, things had improved somewhat but the number
of laboratories able to sequences a hundred thousand
bases was still few
• Not only that, but the cost of sequencing itself was
extremely high and impractical
• Fortunately, there have been vast improvements since
then, particularly in terms of technological advancement
• Better still, automation has made the process much faster daddae?
and a great deal more practical INTRODUCTION
• Now, individual genes are sequenced on a regular basis • It is a method to find out the nucleotide sequence of
and can be done quickly and affordably for laboratories unknown DNA strand
• In fact, some laboratories are sequencing more than a • More recently, Sanger sequencing has been upgraded as
hundred million bases in a given year (Compared back to “Next Generation” sequencing methods, especially for
1970’s nga gamay ra kaayo ilang ma sequence, karon large scale genome analyses and for obtaining especially
they are already sequencing more than 100 million long DNA sequence reads (>500 nucleotides)
bases)
BASIC PRINCIPLE
HOW IS DNA SEQUENCING PERFORMED? • This method generally is an In-Vitro synthesis of DNA
• DNA sequencing involves the process of figuring out the strand, and by using terminators (di-deoxynucleotide) the
precise order of the four bases found in one piece of DNA growing strand terminates at specific sites
• The DNA is really just a template that is used to create a
series of fragments (Di-deoxynucleotides - are chain-elongating inhibitors
of DNA polymerase and they are used to terminate the
• The fragments differ in length by one base, and they are
growing DNA chain and create the subsets of truncated
separated by size before the bases are identified, which
fragments in a sequencing reaction)
then effectively recreates the original DNA sequence
• Each person has twenty-three (23) pairs of chromosomes
• Upon termination, the strands are overlapped to get
- one copy of the human genome
original sequence of unknown DNA strand
• Because technology has limitations, we are limited in how
many bases can be read at one time
• Therefore, we can’t just read each base from one end of
a chromosome to the other. To make it feasible, the
chromosome is cut down into smaller fragments

DIFFERENT METHODS FOR DNA SEQUENCING

Basic Methods
• Maxam-Gilbert sequencing
• Chain-termination methods a. k. a. the Sanger method
Next-generation Methods
In this reaction ddATP is used or utilized. There are actually
• Massively parallel signature sequencing (MPSS) four different types of ddNTPs and ddATP is one of them.
• Polony sequencing
• ddATP – Adenosine nucleotide
• 454 pyrosequencing
• Illumina (Solexa) sequencing
• ddCTP – Cytosine nucleotide

• SOLiD sequencing
• ddTTP – Thymine nucleotide

• Ion Torrent semiconductor sequencing

• ddGTP – Guanine nucleotide

• DNA nanoball sequencing For every nucleotide, there is a corresponding ddNTP. So, in
this case ddATP is being used or d deoxy adenosine 5 prime
• Single molecule real time (SMRT) sequencing
triphosphate.
REQUIREMENTS
SANGER SEQUENCING OR CHAIN TERMINATION
METHOD • Single-stranded template
HISTORY • Primer
• Developed by Frederick Sanger and colleagues in 1977 • DNA polymerase
• It was most widely used sequencing method for • Di-Deoxynucleotide (ddNTP)
approximately 25 years after its discovery o The 3’-OH group necessary for formation of the
• He got NOBEL PRIZE in 1980 phosphodiester bond is missing in ddNTPs

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o Every nucleotide has its specific ddNTP form: • Maxam-Gilbert sequencing was the first widely adopted
ddATP, ddTTp, ddCTP, and ddGTP method for DNA sequencing (along with the Sanger
o “d deoxy (nucleotide) 5 prime triphosphate” dideoxy method)
▪ Adenosine • Method based on chemical modification of DNA and
▪ Thymidine subsequent cleavage at specific nitrogenous bases
▪ Cytidine
▪ Guanosine PRINCIPLE
• Purification of the DNA fragment that is to be sequenced
and labeled with radioactive material
• Chemical treatment generates breaks at specific
nitrogenous bases and thus a series of labelled fragments
is generated. The fragments in the four reactions are
PROCEDURE: arranged side by side in gel electrophoresis for size
• Steps: separation
o Denaturation • The fragments are visualized in X-ray for
o Primer attachment and extension of bases autoradiography
o Termination o Autoradiography is a method used to observe
o Gel electrophoresis a specific site of a biological specimen labeled
• The DNA template is treated with heat so that it becomes with a substance containing a radioactive
single stranded isotope.
• A short, single-stranded primer which is radioactively • To visualize the fragments, the gel is exposed to X-ray
labelled is added to the end of the DNA template film for autoradiography yielding a series of dark bands
• Add template DNA and primer in 4 tubes each corresponding to a radiolabeled DNA fragment,
• Now add ddNTPs to the 4 tubes wherein a single tube from which the sequence may be inferred
contains only one type of ddNTP
PROCEDURE
• Extension starts and fragments formed of various sizes
appear • Maxam-Gilbert sequencing requires radioactive labeling
at one 5’ end of the DNA fragment to be sequenced
• The fragments of DNA are separated by electrophoresis
(gamma-32P)
• Overlap these sequences to find out sequence of target
• Chemical treatment generates breaks at a small
DNA
proportion of one or two of the four nucleotide bases in
each of four reactions (G, A+G, C, C+T). For example:
1. The purines (A+G) by using formic acid
2. The guanines (and to some extent the adenines)
by dimethyl sulfate
3. The pyrimidines (C+T) by using hydrazine
4. NaCl add to hydrazine for Cytosine
• Add each chemical in separate tube
• Thus, a series of labeled fragments is generated
• The fragments in the four reactions are electrophoresed
side by side for size separation
• To visualize the fragments, the gel is exposed to X-ray
film for autoradiography, yielding a series of dark bands
each showing the location of identical radiolabeled DNA
molecules
In this illustration, you can see the four separate tubes with
the single stranded DNA as well as the different DDNTPs
added. All these tubes are then added to the sequencing gel
or your electrophoresis slab/plate to undergo electrophoresis.
Small fragments of DNA will travel farther down the plate while
the larger fragments will remain in the top part of the plate.
The sequences of DNA will then be red starting from the
bottom then going up. Once reading is completed, your
Deduced DNA sequence is then obtained.
MAXAM-GILBERT SEQUENCING METHOD
INTRODUCTION
• Maxam-Gilbert sequencing is a method of DNA
sequencing developed by Allan Maxam and Walter
Gilbert in 1976-1977

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In this example, we have the sequencing of an oligonucleotide • DNA testing has now become routine and expected in
(ONA) by the Maxam-Gilbert method. First, you have your disaster victim identification in the event of a plane
sample ONA, and it undergoes preparation and radio labeling crash, large fire, or terrorist attack
or the addition of your Gamma-32P as your 5’ phosphate. • Dental records and X-rays along with fingerprints are
Afterwards, you will have it undergo cleavage or the breaking normally primarily used in victim identification
at specific nucleotides. The four different reactions with the
• A DNA fingerprint is identical for every part of your body,
different reagents in the four separate tubes consist of your G
whether it is your brain, kidney, or foot. It cannot be
reaction, A+G reaction, T+C reaction, and the C reaction.
changed, so it will be identical no matter what is done to
a body
APPLICATIONS
• The chance of a DNA match between two persons who
• With its study we can understand the function of a specific
aren't twins is from 1/7000 to 1/1,000,000,000,
sequence and the sequence responsible for any disease
depending on the frequency of the patterns being
• With the help of comparative DNA sequence study we
compared
can detect any mutation
• This is a much more specific test than other methods
• DNA fingerprinting
such as blood type, and DNA is present in any kind of
• By knowing the whole genome sequence, Human body tissue, so it is more likely to be found at a crime
genome project was completed. scene than blood
The Human genome project was an international
• DNA testing is also more reliable than eyewitness
collaborative research program with the goal of the
testimony
complete mapping and understanding of all the
genes of human beings
DISADVANTAGES
Forensics • One key disadvantage of DNA analysis is the potential
• DNA sequencing has been applied in forensics science for invasion of individual privacy
to identify particular individual because every individual • Because a person’s DNA reveals so much information
has a unique sequence of his/her DNA. It is particularly about their physical state, it is sensitive information that
used to identify the criminals by finding some proof from must be carefully guarded
the crime scene in the form of hair, nail, skin, or blood • Information about an individual’s ethnic background and
samples. parentage could become cause for discrimination
Agriculture • Disadvantages include incomplete coverage, which can
• DNA sequencing has played a vital role in the field of lead to false normal results, and the ability to test only for
agriculture. The mapping and sequencing of the whole unbalanced rearrangements (duplications and deletions),
genome of microorganisms has allowed the agriculturists and not balanced translocations or inversions
to make them useful for the crops and food plants
CONCLUSIONS
Medicine
• DNA is present in each of our cells and contains the
• In medical research, DNA sequencing can be used to
instructions that allow our bodies to function
detect the genes which are associated with some heredity
or acquired diseases. Scientists use different techniques • Each of our DNA patterns are different, just as our bodies
of genetic engineering like gene therapy to identify the differ. The only exception to this rule is identical twins
defected genes and replace them with the healthy ones • Criminologists can use DNA present at a crime scene to
determine who was present when the crime was
ADVANTAGES committed by comparing these patterns
• One major application of DNA testing is in forensic • While there are several benefits in using DNA analysis to
identification solve crimes, there are still some drawbacks that must be
• DNA test results are much clearer than fingerprints and it considered
is with these results and proof that it is possible to find
criminals Shararawtttt to Mikhael Subaldo for the helping hand!!
• DNA evidence from blood, skin, or hair can be matched thanks miksssss hahaha
to the DNA of a suspect to determine information about
where an individual was and who they may have come in
contact with
• DNA analysis is especially important in cases of rape,
where doctors can often examine a victim and find traces
of the rapist’s DNA
• More and more old crimes are being solved by
resubmitting evidence for enhanced DNA testing
• Another major advantage of DNA analysis is the ability to
screen for certain genetic diseases or risk factors
• Women involved in certain fertility treatments can also get
information about an embryo before it is implanted

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