Sanger Sequencing

Date: 23-11-2023
The human genome project
• Commenced in 1990 - completed in April
2001/2003
• First genome cost $2.7 billion in total
• First time the human genetic blueprint had
been completely ‘read’
Enabled the discovery of genes which
cause disease
Allowed genetic diagnostics to progress

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 History Of DNA Sequencing
DNA ISOLATION DNA Structure 1953
1869

Nanopore sequencing

https://www.nature.com/articles/s41576-020-0275-3

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Sanger Sequencing

• In 1 970s, Frederick Sanger

developed a method for
sequencing DNA that
revolutionized genetics
research
• Sanger sequencing allowed
scientists to read the genetic
code and understand how it
works

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DNA Sequencing
The process of finding the
arrangement/sequence of

Adenine

Thymine

Guanine

Cytosine

https://www.science.org/doi/10.1126/science.1058040

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Difference between deoxy and Dideoxy-
Nucleotides

Nitrogenous
Nitrogenous base
base
P P
5’ 5’
4’ 1’
4’ 1’ Sugar
Sugar
3’ 2’
3’ 2’
OH H H H
Deoxy-nucleotide Di-deoxy-nucleotide

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Principle of Sanger Sequencing
• Sanger sequencing method
is based on chain
termination by the use of
Principle Dideoxynucleotides
(ddNTPs)

• ddNTP is an artificial
molecule that lacks a
hydroxyl group at the 3'
carbon of the ribose sugar
thus terminate the addition
of new nucleotides

https://journals.sagepub.com/doi/full/10.1177/1040638720905833

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Sanger sequencing reactions
For given template DNA, it’s like PCR except:
Uses only a single primer and polymerase to make new
ssDNA pieces
Includes regular nucleotides (A, C, G, T) for extension,
but also includes dideoxy nucleotides

Regular Nucleotides Dideoxy Nucleotides

G A T G A T
G A G C
T C
C C T C A
G A T G A T G
A G G A G
T T A T T A
C C G
A C C A 1. Labeled
T A 2. Terminators
C G

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DiDeoxy-Nucleotides causing chain
termination

https://www.google.com/url?sa=i&url=https%3A%2F%2Fen.wikipedia.org%2Fwiki
%2FSanger_sequencing&psig=AOvVaw2Kkg8epNCHnkNyekgdoTHm&ust=1694707336178000&source=images&cd=vfe&o
pi=89978449&ved=0CAQQjB1qFwoTCMjCj_v6p4EDFQAAAAAdAAAAABBk

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Principle of Sanger sequencing- Chain
termination

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 Illustration of conventional Sanger
Sequencing process

Denaturation of
synthesized chains
to single strands

Addition of Polymerization
Amplification primer to Addition of
of single continues until
initiate dNTPs and
Denaturation of DS stranded incorporation
polymerizati ddNTPs Fragments are
DNA into SS DNA target DNA of ddNTP
on of DNA separated by
electrophoresis and
sequence is read.

https://www.researchgate.net/figure/The-Sanger-sequencing-method-in-7-steps-1-The-dsDNA-fragment-is-denatured-into-two_fig2_234248746

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Automated version of the dideoxy
method

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Automation of sequencing process
First automated sequence
was invented by Lyod.
M.Smith, in 1987

It uses Sangers sequence

method

AB370A was able to

sequence 96 samples
simultaneously with 500-
600kb in size AB310- First automated sequencer
developed by Applied Bio-systems utilized
in the ‘’human genome project’’ in 2001.

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Sequencing results
Chromatogram: Each sequencing reaction gives us
a chromatogram, usually ~600-1000 bp

https://www.labxchange.org/library/items/lb:LabXchange:22c08d85:html:1

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Sequencing results

https://www.weizmann.ac.il/LS_CoreFacilities/dna-sequencing/results-analysis

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DNA Template Quality
Poor template quality is the most common cause of
sequencing problems
Results characteristic of using poor quality templates:
• Noisy data or peaks under peaks
• No or low signal
• Early loss or termination of extension

15
16
Advantages
Not as toxic and less radioactivity than Maxam and Gilbert
method

Easier to automate-This lowered the cost from $100 million to

$10,000 USD in 2011

Highly accurate long sequence reads of about 700 base pairs

Easier to get started – by using commercially available kits

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Applications of DNA sequencing
DNA sequencing has numerous
applications in fields such as
medicine, forensics,
agriculture, and evolutionary
biology
It is used for disease diagnosis,
genetic counseling, criminal
investigations, crop
improvement, and phylogenetic
analysis

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Applications in medicine
Sanger sequencing has
numerous applications in
medicine, including
• Diagnosing genetic disorders,
• Identifying disease-causing
mutations
• Developing personalized
treatments
• It has also led to
breakthroughs in cancer
research

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 Applications in research
• De novo sequencing of genomes
• Detection of variants (SNPs)
and mutations
• Biological identification
• Confirmation of clone
constructs
• Detection of methylation events
• Gene expression studies
• Detection of copy number
variation

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Limitations

While Sanger sequencing is a

powerful tool, it does have
limitations

It is not suitable for sequencing

large genomes

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DNA Template Preparation
Workflow
Isolate DNA from source i.e cells

Amplify DNA template by PCR

Design primers

Clean up templates

Examine DNA quality

Determine DNA quantity

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Length of Genomic DNA Fragments

Genomic DNA fragments longer than 50 kbp may be less

efficient targets for PCR amplification
Isolating long DNA fragments, may need to shear the DNA by
• Vortexing
• Passing it through a needle

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Minimizing Contaminants
• Amount of starting DNA
• Careful primer design
• Primer concentration
• Enzyme concentration
• Magnesium ion (Mg2+) concentration
• Nucleotide concentration
• Buffer composition
• Number of cycles
• pH
• Manual hot-start method

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Purifying PCR Products for
Sequencing

There are several

methods for purifying
PCR products

Ethanol Gel Enzymatic

Ultrafiltration precipitation purification purification

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Examining DNA Quality
Agarose gel electrophoresis
Purified DNA should run as a single, intact band
Spectrophotometry
The A260/A280 ratio should be 1.8 to 2.0.
NanoDrop
Measure the concentration of DNA
Thanks-------