DNA SEQUENCING METHODS

Presented by-
Dipesh Kumar Karn
Roll No-10
B.Pharm. Second year, Third Semester
School of Health and Allied Sciences, Pokhara
University, Pokhara-30 Kaski
 OUTLINE
1. INTRODUCTION
2. METHODS OF DNA SEQUENCING
3. SANGER SEQUENCING METHODS
4. APPLICATIONS OF DNA SEQUENCING
5. CONCLUSION
 INTRODUCTION
• The information content of DNA is encoded
the form of four bases (A,G,T and C) and the
process of determining the sequences of
these bases in a given DNA molecule is
called DNA sequencing.
• DNA sequencing is the process of
determining the nucleic acid sequence –the
order of four nucleotides in DNA.
• DNA fragments can be analyzed to determine
the nucleotides sequence of DNA and to
determine the distribution and location of
restriction sites.
 METHODS
1. Basic DNA sequencing
a. Sanger method of DNA sequencing (Chain Termination method)
b. Maxam-Gilbert method of DNA sequencing(Chemical termination method)
2. Advanced DNA sequencing
Shotgun sequencing
3. Next generation DNA sequencing
-Solid sequencing
- Ilumina sequencing
-Pyrosequencing

Here, I am going to describe about sanger sequencing method.

 SANGER SEQUENCING
• Sanger sequencing is a method of DNA sequencing that involves electrophoresis
and is based on the random incorporation of chain
terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
• It was first developed by Frederick Sanger and colleagues in 1977 and it became
the most widely used sequencing method for approximately 40 years.
• It was first commercialized by Applied Biosystems in 1986. More recently, higher
volume Sanger sequencing has been replaced by next generation sequencing
methods, especially for large-scale, automated genome analyses.
• However, the Sanger method remains in wide use for smaller-scale projects and
for validation of deep sequencing results.
 PROCESS
In this method we need following things;-
I. A labelled DNA primer
II. DNA polymerase
III.4 types of deoxriboynucleosides 5’ triphpsphates
(dATP, dGTP, dTTP, dCTP)
IV.A tiny quantity of specific dideoxynucleotide
triphosphate
 STEPS
1)The double stranded DNA molecule to be sequenced is denatured in a solution of
NaOH.
2) The solution containing single strand of DNA is mixed with following;-
a. A labelled DNA primer (complementary to 3’ end of the strand
to be sequenced)
b. DNA polymerase
c. dATP, dGTP, dCTP, dTTP
d. A tiny quantity of ddNTPS
 CONTD….

3) Step 2 is repeated three different times with the other three different ddNTPS.

4)Once the four reactions are completed, we run gel electrophoresis.

Each mixture is placed into a lane to produce a total of four lanes. The results are
transferred onto a polymer sheets which is then exposed to x-ray autoradiography.
This tells us exactly where the radioactively labelled fragments are.
In gel electrophoresis, the fragments that migrated the farthest are the smallest.
NaOH
ddATP ddGTP ddCTP ddTTP

C
G
T
A
A

T
 APPLICATION OF DNA SEQUENCING
a. Understanding the function of a specific sequence and the sequence
responsible for diseases.
b. With the help of comparative DNA sequence study, we can detect any
mutation.
c. Helps in DNA fingerprinting.
d. In forensics- DNA sequencing has been applied in forensics science to
identify particular individual because each individual has unique
sequence of his/her DNA .
e. Ultimately, DNA sequencing will become a part of a patient’s medical
record, helping physicians to determine the patient’s risk of certain
disease and optimal treatments.
 CONCLUSION
DNA sequencing is one of the important process which provides the means
to know how nucleotide bases are arranged in a piece of DNA. It has for
example provided an important tool for determining the thousands of
nucleotide variations associated with specific genetic diseases like
Huntigton’s, which may help better understand these diseases and
advance treatment. DNA sequencing also underpins pharmacogenomics,
which looks at how a person’s individual genome variation affect their
response to drugs.
 REFERENCES
• https://en.wikipedia.org/wiki/DNA_sequencing
• https://www.slideshare.net/mallappashalavadi/dna-sequencing-
18897579
• https://www.youtube.com/watch?v=I5pwZTJMVCQ
• https://aklectures.com/lecture/biotechnology-and-recombinant-dna-
technology/sanger-sequencing-of-dna