COMSATS Institute of Information Technology

BS Course (BIO346)
Introduction to Genomic and
Proteomics

Dr. Rabia Habib

Assistant Professor
Department of Biosciences
DNA Sequencing
Methodologies
DNA sequencing refers to sequencing methods
for determining the order of the nucleotide
/bases in a DNA molecule.
• The DNA seq. techniques in use today can be
divided into two categories:

 The chain-termination method/Sanger sequencing:

which was first devised by Fred Sanger and
colleagues in the mid-1970s

 Next-generation sequencing:
which is a collection of methods, each of which
utilizes a massively parallel strategy in order to
generate millions of sequences at the same time
 DNA Sequencing Methods
1. Chain Termination Reaction or Dideoxy
/Sanger Sequencing
2. Pyrosequencing
SANGER /DIDEOXY SEQUENCING
 Sanger /Dideoxy-Terminating
Sequencing
 DNA sequencing based on the selective
incorporation of chain-terminating
dideoxynucleotides (ddNTPs) by DNA
polymerase during in vitro DNA synthesis.

 Method was invented by Frederick

Sanger in 1977

 Sanger Seq. became the method of choice

due to ease and reliability.

 500-900bp reads per reactions obtainable

The dideoxy method of DNA sequencing is based on the
termination of DNA synthesis
 Component of the Reaction
Mixture
•The Sequencing reaction mixture contains:
•Single-stranded DNA template to be sequenced,
• Specific Primer
•DNA Polymerase,
• All four dNTPs (dATPs, dTTPs, dGTPs,dCTPs),
•Small amount of each of four ddNTPs (ddATP,
ddTTP, ddGTP, ddCTP), each labeled with
different fluorescent markers.
 1. DNA Synthesis: Primer Annealing

2. Chain Extension and Termination

3. Polyacrylaminde/urea gel
electrophoresis and Fluorescence
detection
 The dideoxy sequencing method :

1. Each of the four ddNTPs is

tagged with a fluorescent dye

3. The fluorescent dye on

the DNA is detected by a
laser beam

4. The sequence
information is read directly
2. Denatured products and stored electronically
produced loaded into a single into the computer which
well on an electrophoresis gel. converts it into the
The fragments migrate through complementary target
the gel according to size,… sequence.
DNA sequencing printout, represented by
a series of peaks: Chromatogram
Nucleotide Sequence

Number of Nucleotide
5’ 3’
 Thermal Cycle Sequencing
• PCR is carried out with just one
primer and with the labeled four
ddNTPs present in the reaction
mixture.
• The result is a set of chain-
terminated strands which are
electrophorised using standard
sequencing methodology.

• Advantages over traditional chain

termination sequencing.
1. uses double-stranded rather than
single-stranded DNA as the starting
material.
 Polyacrylamide gel electrophoresis (PAGE)

• Separation of DNA molecules differing in length by just

one nucleotide (10 to 1500 bp in length)
Gel Components:
 Chains of acrylamide monomers cross-linked with N, N’-
methylenebisacrylamide units (referred as ‘bis’)
 TEMED (N, N, N’, N’-Tetramethylethylenediamine)
catalyst for polymerization reaction
 Ammonium persulfate act as activator

 Urea added as denaturant for preventing intrastrands

basepairs formation
Polyacrylamide gel electrophoresis in a capillary system
can resolve single-stranded DNA molecules that differ in
length by just one nucleotide.
 Capillary Gel Electrophoresis

Fluorescence
detector

Laser beam

Schematic of system. Samples enter the tube from one end and travel to the left to
the detection system which records the chromatogram output on a computer
Pyrosequencing
• ‘’Sequencing by synthesis’’
• No need for gel electrophoresis
• Order of nucleotide incorporation is detected by
pyrophosphate (PPi) release
• Pyrophosphate converted by the enzymes sulfurylase
and luciferase into a flash of chemiluminescence
recorded as peaks on a pyrogram.
• The amount of light emitted is proportional to number
dNTPs incorporated into the growing strand

• Easily automated (up to 1.6 million reactions can be

carried out in parallel on a 6.4 cm2 slide = 25 millionbp
in 4hr)
Pyrosequencing Results: Pyrogram
Nucleotide Sequence

Nucleotide Added

5’AGGGGTGGCTTTGGGG…………….3’