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Sanger Sequencing

DNA sequencing is the process of determining the order of nucleotides in DNA. Sanger sequencing, developed by Frederick Sanger in 1977, uses a chain termination method to identify nucleotide sequences, involving steps such as denaturing DNA, adding primers, and using polymerase with dNTPs and ddNTPs. The method can be performed using either the chain termination method or dye-terminator sequencing, which utilizes fluorescent dyes for detection.

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Shivi Chauhan
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8 views12 pages

Sanger Sequencing

DNA sequencing is the process of determining the order of nucleotides in DNA. Sanger sequencing, developed by Frederick Sanger in 1977, uses a chain termination method to identify nucleotide sequences, involving steps such as denaturing DNA, adding primers, and using polymerase with dNTPs and ddNTPs. The method can be performed using either the chain termination method or dye-terminator sequencing, which utilizes fluorescent dyes for detection.

Uploaded by

Shivi Chauhan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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DNA

SEQUENCIN
G METHODS
Made by: Vibhuti R Jakhmola
Sujata
Bhattacharya
Shivi Chauhan
What is DNA Sequencing?
DNA sequencing is the process of determining
the nucleic acid sequence – 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.
Sanger
Sequencing
Sanger sequencing, also known as the
“chain termination method,” was
developed by the English biochemist
Frederick Sanger and his colleagues in
1977.
This method is designed for determining
the sequence of nucleotide bases in a
piece of DNA (commonly less than 1,000
bp in length).
DNA Polymerase Enzyme

Requirements A Primer

for Sanger ddATP, ddGTP, ddTTP, ddCTP,


Sequencing each labelled with a different
colour dye.

DNA Template to be sequenced


Principle
In Sanger sequencing, a DNA primer complementary to the
template DNA (the DNA to be sequenced) is used to be a
starting point for DNA synthesis. In the presence of the four
deoxynucleotide triphosphates (dNTPs: A, G, C, and T), the
polymerase extends the primer by adding the
complementary dNTP to the template DNA strand.
Compared to dNTPs, ddNTPs has an oxygen atom removed
from the ribonucleotide, hence cannot form a link with the
next nucleotide.
Steps in Sanger Sequencing
The Sanger sequencing method consists of 6 steps:
(1) The double-stranded DNA (dsDNA) is denatured into two
single-stranded DNA (ssDNA).
(2) A primer that corresponds to one end of the sequence is
attached.
(3) Four polymerase solutions with four types of dNTPs but
only one type of ddNTP are added.
(4) The DNA synthesis reaction initiates and the chain extends
until a termination nucleotide is randomly incorporated.
(5) The resulting DNA fragments are denatured into ssDNA.
(6) The denatured fragments are separated by gel
electrophoresis and the sequence is determined.
Method

CHAIN TERMINATION DYE-TERMINATOR


METHOD SEQUENCING
The DNA sample is divided into four separate
sequencing reactions, containing all four of the In dye-terminator sequencing, each of the
standard deoxynucleotides (dATP, dGTP, dCTP four dideoxynucleotide chain terminators is
and dTTP) and the DNA polymerase. To each labelled with fluorescent dyes, each of which
reaction is added only one of the four emits light at different wavelengths.
dideoxynucleotides (ddATP, ddGTP, ddCTP, or
ddTTP), while the other added nucleotides are
ordinary ones.
THANK YOU!

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