DNA SEQUENCING
Presented to:
DR. HAFIZ ABDULLAH
SHAKIR
Abdullah
MS-UZE06S22
Judges:
Abdul manan (MS-08)
Fazah Ali (MS-04)
Ayesha khan (MS-05)
2
Outlines
1 Introduction
2 Methods
Basic:
1 Maxam Gilbert Method
2 The Sanger’s Method
Advanced:
3 Shotgun Sequencing
Methods
Next Generation:
4 Automated Florescence sequencing
5 Pyrosequencing
3 Applications
3
INTRODUCTION
DNA sequencing is the process of determining the nucleic acid
sequence and the order of nucleotides in DNA.
It includes any method or technology that is used to determine the
order of the four bases: adenine, guanine, cytosine, and thymine .
NITROGENOUS BASES
THE PURINES IN DNA ARE ADENINE AND GUANINE
THE PYRIMIDINES IN DNA ARE CYTOSINE AND THYMINE
Watson and Crick structure of DNA
Watson and Crick proposed that the DNA is made up of two
strands that are twisted around each other to form a right-
handed helix, called a double helix.
Base-pairing takes place between a purine and pyrimidine:
namely, A pairs with T, and G pairs with C.
Basic DNA Sequencing Methods
1 Maxam and Gilbert method
2 Sanger method
1. Maxam Gilbert Method
• Maxam–Gilbert sequencing is a method of DNA sequencing
developed by Allan Maxam and Walter Gilbert in 1976–1977.
• This method is based on chemical modification of DNA and
subsequent cleavage of the DNA at specific desired
nucleotides
Steps
labelling at 5’ chemical cleavage
end treatment
electrophoresis
reading from and
bottom to top autoradiography
1. Labelling of DNA
• Multiple copies of the sample DNA are produced
• Remove the phosphate at the 5’ end and incorporate the radioactive
phosphate 32 PO4 enzymatically.
2. Chemical treatment
3. Cleavage
4. Gel Electrophoresis
• 4 samples and one ladder (known sequence) are placed in wells on the
gel
• Fragments would separate on gel
according to size
• smaller fragments would move
farther than larger fragments
• After placing radioactive film on
the gel radioactive labelled
fragments would emit a spot at
their position
[Link] from bottom to top
We obtain the sequence by reading from bottom to top.
Advantages
• Directly read purified DNA
• used to analyze the protein and DNA interaction
Disadvantages
• use of toxic chemicals and radioactive isotopes
• setup is quote complex
• It is difficult to make Maxam-Gilbert DNA sequencing kit
• Cannot read more than 500 base pairs
2. The Sanger’s Method of Sequencing
Developed by:
Fredrick Sanger in 1977
Principle :
Sanger sequencing is a method of DNA sequencing that is
based on the random incorporation of chain-terminating
dideoxynucleotides by DNA polymerase during in vitro DNA
replication.
If during replication ddNTPs is incorporated instead of usual
dNTPs in the growing DNA strand then the replication stops
at that nucleotide.
Deoxynucleotide
DNA polymerase requires 3’ OH group and 5’ phosphate group of
incoming dNTP to create a phosphodiester bond
Bonding between Nucleotides
Conti…
Conti…
Conti…
Phosphodiester Bond
Dideoxynucleotide
3’ OH group is absent responsible for the chain elongation
Conti…
ddNTPs may be radioactively or fluorescently labelled
for detection in automated sequencing machines.
Steps
1. Mix reagents
2. Primer attached and extension of bases
3. Chain Termination
4. Polyacrylamide gel electrophoresis
Mix reagents in 4 separate
tubes
Tube 1
• Contains ddATP
• Each tube has a primer, dNTPs (A, T, G and C) DNA polymerase
Adding nucleotides to the
growing chain
Chain termination
Tube 1
ddATP
Sequencing
Autoradiography
After autoradiography, the nucleotide sequence of desired DNA
fragment is obtained
Limitations
• Only sequence short pieces of DNA i.e., 300-1000 bp
• If the DNA fragment being sequenced has been cloned, some of the
cloning vector sequence may find its way into the final sequence.
Sanger sequencing is a good
choice when:
• Sequencing single genes
• Analyzing fragments
• Sequencing 96 samples or less
3. Shotgun Sequencing
Developed by:
Fred Sanger and his colleagues in 1979
Principle:
DNA is broken up randomly into numerous small fragments and
overlapping regions are identified between all the individual
sequences.
Multiple overlapping reads for the target DNA are obtained by
performing several rounds of this fragmentation and sequencing.
It was the initiative for full genome sequencing.
Enzyme based Acoustic shearing
treatments
DNA
SHEARING
Centrifugal Sonication
shearing
PROCEDURE
Faster process Requires large amount of
Less expensive computing power and
sophisticated software
Repetitive genomes and
sequences are difficult to be
assembled
Next generation method
• Basic methods are expensive and time consuming. They are not so fast to sequence
an entire genome of an organism.
• When the human genome project was launched it assume to take 15 years to
complete because at that time all the known methods were basic.
• Most of the next generation methods make the use of fluorescent nucleotide or
other molecules produced during the replication process.
4. Automated Florescence sequencing
• In the modified form of sanger method, the fluorescent Dideoxy
nucleotides are added and after the generation of fragments the
fragments are run into a tube having the laser and a detector at the
lower end when fragments run in the tube the fragment with smallest
length reaches first than gradually with increasing length cross the laser
and produce their respective colour and detected by detector each
colour is associated with specific nucleotide.
• It makes the process faster. First a colour pattern is produced, and it is
coded into the complementary nucleotide sequence which is then
changed into the original DNA sequence
5. Pyrosequencing
• In this method the tri-phosphate nucleotide is added.
• when this is added to in replication process it produces a pyrophosphate (ppi) which on
the addition of some chemical make the ATP.
• Luciferin and luciferase enzyme is added which glow on the production of ATP because
enzyme use ATP energy to produce glow. Glow indicate the addition of specific
nucleotide.
• This indication is used to determine DNA sequence.
Application of DNA sequencing
• DNA sequencing has its prominent role in each field of biology and
other sciences like Medicine, forensic and now its role in research work
is increasing.
•It is used to determine the sequence of entire genome of an organism,
a single chromosome or for a single gene.
•There are some fields in which DNA sequencing is used
1. Molecular biology
In molecular biology we use DNA sequencing to read genome and the proteins
encoded by that genome. It gives information to researchers to find the change in gene
related to some disease and phenotype. It helps to target the drug at specific site.
2. Evolutionary Biology
DNA is involved in transmission of all the traits to offspring. It is most advance
and much prominent evidence of evolution to tell how different organisms are related to
each other.
3. Medicine
In medical field it provides evidence of a genetic diseases. It informs about the
presence of a viral disease by finding the genome of that virus in blood sample i.e.,
finding the type of hepatitis virus A, B or C
? & !
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