Seminar:
DNA analysis
Sanger sequencing
OUR TEAM
Trương Nguyễn Nghiêm Gia Hồ Học Nghi
Hoàng Long Phụng
Student ID 207720201024 Student ID 207720201030 Student ID 207720201026
01 02
INTRODUCTIO
N PRINCIPLE
03 04
CONCLUSI
PROCEDURE-
MODERN ON
PREPARATION
01 INTRODUCTION
Important of DNA
sequencing
➢ DNA sequence information is important to scientists
investigating the functions of genes.
➢ Providing insights into the pathogenesis of human
diseases.
History
➢ First developed by Frederick Sanger and
colleagues in 1977,
➢ Became most widely used sequencing
method for 40 years.
➢ First commercialized by Applied
Biosystems in 1986.
Frederick Sanger
History
In 1977,
- Sanger and Colleagues -
Publication of DNA sequencing with
chain-termination inhibitors.
02
PRINCIPLE
- What is sanger sequencing?
- Mechanism
WHAT IS
SANGER
SEQUENCING?
2. PRINCIPLE
What is sanger sequencing?
Sanger sequencing (Chain-termination PCR ) works just like standard PCR, but with
one major difference: the addition of modified nucleotides (dNTPs) called
dideoxyribonucleotides (ddNTPs).
2. PRINCIPLE
Mechanism
Modified nucleotides (dNTPs) called
dideoxyribonucleotides (ddNTPs).
--Lack OH group
--Stop the enlogation process
2. PRINCIPLE
Normal DNA
polymerisation
DNA Pol
2. PRINCIPLE
DNA Pol
2. PRINCIPLE
DNA polymerization
With Addition of
ddNTP
DNA Pol
ddATP
2. PRINCIPLE
DNA polymerization
With Addition of
ddNTP
DNA Pol
Chain termination
2. PRINCIPLE
Mechanism
dGTP, dCTP, dATP, dTTP
DNA pol, primer,
ddTTP ( small amount ) 3' --------- A-G-T-G-T-A-T-C-G-A 5'
buffer,…
3' --------- A-G-T-G-T-A-T-C-G-A 5'
5' Primer-T-C-A-C-A- T 3'
3' --------- A-G-T-G-T-A-T-C-G-A 5'
5' Primer-T-C-A-C-A-T-A-G-C- T 3'
→ Millions to billions of oligonucleotide copies of
the DNA sequence of interest, terminated at random
3' --------- A-G-T-G-T-A-T-C-G-A 5'
5' Primer-T 3'
lengths (n) by 5’-ddTTPs.
2. PRINCIPLE
Mechanism 4 reactions: each reaction add
only 1 type of ddNTP
dGTP, dCTP, dATP, dTTP
ddTTP ( small amount )
3' --------- A-G-T-G-T-A-T-C-G-A 5'
5' Primer-T-C-A-C-A- T 3'
3' --------- A-G-T-G-T-A-T-C-G-A 5'
5' Primer-T-C-A-C-A-T-A-G-C- T 3'
3' --------- A-G-T-G-T-A-T-C-G-A 5'
5' Primer-T 3'
2. PRINCIPLE
Mechanism
3' --------- A-G-T-G-T-A-T-C-G-A 5'
ddTTP ddGTP
→ Millions to billions
of oligonucleotide
copies of the DNA
sequence of interest,
ddATP ddCTP terminated at random
lengths (n) by 5’-
ddNTPs.
2. PRINCIPLE
Mechanism
Chain-termination PCR
The chain-terminated oligonucleotides are separated by size via gel
electrophoresis.
1, The negative charge of its phosphate backbone moves the DNA towards the
positive charged anode
2, Shorter DNA molecules can travel farther than longer counterparts
2. PRINCIPLE
Mechanism
Electrophoresis
-Separated by size
3' --------- A-G-T-G-T-A-T-C-G-A 5'
ddTTP
ddCTP
ddATP
ddGTP
2. PRINCIPLE
Mechanism
Chain-termination PCR
Reading the gel to determine the sequence of the input DNA.
Each terminal ddNTP will correspond to a specific nucleotide in the original
sequence → By reading the gel bands from smallest to largest, we can
determine the 5’ to 3’ sequence of the original DNA strand.
2. PRINCIPLE Chain-termination PCR
Mechanism Reading the gel bands from smallest to largest
This is from 5’ to 3’ direction of the elongation chain
-
5’ Primer-T-C-A-C-A-T-A-G-C-T 3’
3’ ----------A-G-T-G-T-A-T-C-G-A 5’
+
2. PRINCIPLE
Mechanism
-Fluorescence dye help better visualizing DNA
2. PRINCIPLE
In summary, The steps for Sanger sequencing
ddATP
Polymerisation with ddTTP Size separation by
addition of small amount Electropherosis
All possible fragments
of 1 type ddNTP (with visualization)
ddGTP
ddCTP
03
PROCEDURE - MODERN
PREPARATION
3. PROCEDURE - MODERN PREPARATION
3. PROCEDURE - MODERN PREPARATION
Polymerase chain reaction (PCR) is a technique used to amplify a single copy or
a few copies of a piece of DNA across several orders of magnitude, generating
thousands to millions of copies of a particular DNA sequence.
An overview of the PCR
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
1. Template preparation (Plasmid ,PCR… template )
- DNA extraction
- PCR Amplification – with primer design
- PCR Cleanup
2. Cycle sequencing
- Sequencing cleanup
3. Data/DNA visualizing
- Capillary electrophoresis
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
1. Template preparation
DNA extraction
-- Select the right method/kit for the sample type
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
1. Template preparation
Plasmid template
-- Commercial kits or general methods
-- A single colony is used for extraction
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
1. Template preparation
Primer design
-- Produces a strong single product of the correct size
-- Depending on the circumstances → use another approach
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
1. Template preparation
PCR Amplification
The extracted DNA are amplified to multiple copies
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
1. Template preparation
PCR Cleanup
-- Gel purification
-- Enzymatic purification
-- Ethanol purification
-- Column purification
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
1. Template preparation
PCR Cleanup
Process:
-- Treat PCR product with ExoSAP-IT
ExoSAP-IT™ PCR Product Cleanup Reagent is used for enzymatic cleanup
of amplified PCR product.
-- Seal and then spin
-- The samples are incubate
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
2. Cycle sequencing BigDye Terminator version 3.1 (BDTv3.1)
BigDye Terminator version
3.1 ( BDT v3.1 )
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
- Cycle sequencing
Process:
-- Sequencing reactions using the: BigDye Terminator (BDT)v3.1 Ready
Reaction Mix; Deionized water
-- Forward sequencing primer
-- Reverse sequencing primer
-- Run the reactions in a thermal cycler
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
2. Cycle sequencing
Proposed cycle procedure for set up
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
2. Cycle sequencing
Sequencing Cleanup
→ Remove unlabeled and dye-labeled components
Process:
-- Purify the sequencing products by EDTA/EtOH method (with the Bigdye
XTerminator Purification Kit)
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
3. Data/DNA visualizing
- Capillary electrophoresis
In the Past: Autoradiography ( hard to collect data )
Still need human work
Autoradiograph 1977 version
Sanger and colleagues
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
- Capillary electrophoresis
In the Past: Autoradiography ( hard to collect data )
Still need human work
1993 version James
L Van Etten
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
- Capillary electrophoresis
r
t ect o
-Derivative of De
electrophoresis (same - +
mechanism)
r
se
-Detector recognize specific La
wavelength emit from the
labelled DNA fragment
Characterized as
peak DNA sample
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
- Capillary electrophoresis
r
Automatic collecting data t ect o
Characterized as peak De
- +
A G T G T C C A
r
se
La
DNA sample
3. PROCEDURE - MODERN PREPARATION
How to perform sanger sequencing?
- Capillary electrophoresis
Automatic collecting data BigDye Terminator version 3.1
Characterized as peak (BDTv3.1)
04
CONCLUSION
4. CONCLUSION
Advantages
-- Fast, cost-effective sequencing for low numbers
of targets
-- Familiar workflow
-- Sequencing of whole genomes
Disadvantages
-- Low sensitivity
-- Low discovery power
-- Not as cost-effective for high numbers of targets
-- Low scalability
Common questions
How do you know the primer sequence (for PCR) during
sequencing ?
The best thing you can do is search databases (NCBI, Ensembl) for sequences
of the closest relatives you can find.
Find the most conserved part --> design a set of primers, which would account
for all possible nucleotide combinations
However, there's a computer software with database do this for you
Note: Universal primers ( design by companies ) only work with specific
type of template
Common questions
The accuracy? Is the result is from right after the primer?
-- High accuracy if follow procedure – short fragments
-- In fact that high accuracy only after a few base right after the primers
Can directly sequence only relatively short (300-1000 nucleotides long) DNA
fragments in a single reaction.
The main obstacle to sequencing DNA fragments above this size limit is
insufficient power of separation for resolving large DNA fragments differ in
length by only one nucleotide.
--> Reversed primer to ensure the results
REFERENCES
1. Guide to Sanger Sequencing at RAMAC ( Ramacioti
Centre of Genomics )
2. Sanger F, Nicklen S, Coulson AR. DNA sequencing
with chain-terminating inhibitors. Proc Natl Acad Sci U S
A. 1977;74(12):5463-5467. doi:10.1073/pnas.74.12.5463
3. Karger BL, Guttman A. DNA sequencing by
CE. Electrophoresis. 2009;30 Suppl 1(Suppl 1):S196-
S202. doi:10.1002/elps.200900218
Thank you
for your
attention
