Sequencing of DNA

(Sanger Sequencing)

Sirjan Bastola

2019-05-18
 What is ddNTP?
 dNTP = dideoxyribonucleotide triphosphate
 dNTP = dideoxyribose sugar + N-base + triphosphate group
 e.g. ddATP, ddGTP, ddCTP, and ddTTP

Remove O from C-3

OH H

Structure of dNTP Structure of ddNTP

Chain elongating ribonucleotide Chain terminating ribonucleotide

(3’ –OH group is necessary to continue chain)
 Sanger sequencing of DNA
 DNA sequencing is the process of determining the N-bases
(A,T,G,C) in DNA.
 Sanger sequencing is also known as Chain termination
method or Dideoxy technique of DNA sequencing.
 It’s the “First generation” sequencing method .
 Process of selective incorporation of chain-terminating
dideoxynucleotides by DNA polymerase during in vitro DNA
replication.
 Developed by Frederick Sanger
in 1975 and received Nobel Prize
in Chemistry in 1980.
 What happens in Sanger sequencing of DNA?

 Target DNA is copied many times, making fragments

of different lengths.
 Fluorescent “chain terminator” nucleotides mark the
ends of the fragments and allow the sequence to be
determined.
 5 Requirements for Sanger Sequencing of DNA
1. Sample of DNA containing target sequence
2. All 4 Deoxyribonucleotides (dNTP) = dGTP, dCTP, dATP and dTTP
3. Thermostable DNA Polymerase = Taq Polymerase
(Obtained from bacterium Thermus Aquaticus.)
4. Single Primer = Short DNA fragment complementary
to DNA sequence at 3’ end of target sequence
5. All 4 Dideoxyribonucleotides (ddNTP)= ddGTP, ddCTP, ddATP and ddTTP

Each ddNTP is labelled with

a specific colour of dye
(Fluorescent tag/molecule)
 Sanguer Sequencing of DNA

PCR tube
with raw materials

Thermocycler
 Transfer PCR tube to thermocycler
 Set the temperature conditions for amplification of DNA
 Transfer reaction product to sequencing machine
(Product 96 well plate  sequencing machine)
 Run the sequencing machine with help of computer program
and get the raw data on computer after completion of sequence reading
 Analyze the raw data using software to get the real sequence data
Principle of Sanguer Sequencing
 References
 https://
www.khanacademy.org/science/high-school-biology/hs-m
olecular-genetics/hs-biotechnology/a/dna-sequencing
 https://
www.sigmaaldrich.com/technical-documents/articles/biol
ogy/sanger-sequencing.html
 Please watch the you tube video
1)https://
www.youtube.com/watch?v=e2G5zx-OJIw
2)https
://www.youtube.com/watch?v=Wpww8bb
63zU
 Search Assignment

1) Next Generation Sequencing (NGS)

2) Bioinformatics