How does Sanger sequencing work?

Sanger sequencing, or the enzymatiC chann termination method. is based on the se o

fiuorophore-labeled dideoxynucleotides with (ddNTPs)
regulu!
in Combination
deoxynucleotides (dNTPs). Because ddNTPs lack a hydroxyl group needed for nucleotide
binding. the addition of ddNTPS by the DNA polynicrase during chain extension terminates
the DNA strand elongation. Reiteration of primer annealing and DNA extension
cycles results
gments with a tluorophore-labeled nucleotide at each position, thereby identifying every
nucleotide in the DNA template (Crossley et al. 2020).

Reagents (1) Primer anneal1ng and chain extension (2) ddNTP binding and chain terminaticr

dT cctTF dTP

TTTTT TTTTTTT
eSF .

TT T
TITITTTTITTT

3 Fuorescently labelled DNk 3ample (4) Capillary gel electroplhoresis and (5) Sequence analy sis and reconstruciicn
fluorescence deteciion
GAC
TTTTTTTTmTTTITTTTTT DNA Sanple

TTTTTTTTTTTTTTTU

liiTT
TTTTTTTTO3
Detector
TTT

Sanger sequencing method. 1- Deoxymucleotides (NT? and a low concentration of

fluorophore-abeled ideorymucleotides (adedNT?s) ave suyppied 1ogether wiuh DNA tomplate
and primers. Polymerase calalyzes the DNA chain extensio1 by ranlom incorporution of
available nucleotides. 2- Binding ofafluorophore-labelecd ddNTP leads to termination ofchain

extension 3- DNA fragmenis of variotus lengths m labelu 12 i/ervni Huoophores dare

generated. 4- Labeled DNA Jragments are separated bused on noleculur nass by capillarv gel

electrophoresis, and their associated Auorescence is detected hy a Churge-Coupled Device

CCD). 5-A chronnatogram is generated based on peak fluorescece detecied, corresponding

euch position o/ 1hie DN-A egee. Reprinted
tofluorophore-labeled dlNTPs incorporated at
from "Sanger Sequencing". by BioRender.com (2021).
 How does Sanger sequencing work?
Sanger sequencing. or the enzymatic chain temination method. is based on the use ot
fiuorophore-labeled dideoxynucleotides (ddNTPs) in combination with regular
deoxynucleotides (dNTPs). Because ddNTPs lack a hydroxyl group needed for nucleotide
binding. the addition of ddNTPS by the DNA polynmerase during chain extension terminates
the DNA strand elongation. Reiteration of primer annealing and DNA extension eycles results
in fragments with a tluorophore-labeled nucleotide at each position. thereby identifying every
nucleotide in the DNA template (Crossley et al. 2020).

Reagents OPrimer annealing and chain extensan 3dNTP >ndng and chasin terntee

adTTF
Titliif

3 Fuorescenty labeled DN smple Capillan gel etectrcohoresis an Squenre anai, is and rconshciea
fiuorescence deteeivan
iitTiEiTTUTTTUTTTT

TTTITTTTTTTTTTT

Sanger sequencing method. 1- Deorymucleotides (N" nd a io cocenration of

fluorophore-labeleddideorymucleotides (ddNTPs) are sufied togetiher wiuh DN.t Iemplate
und primers. Polymerase caialyzes the DNA chuin ertensi:n! by rarndom incorporatien of
available nucleotides 2- Binding of a tluoropkore-labelecd diNTP 1eeds to ternitiition of chain
exriension3- DNA fragmenmsofvw ious lengths m. ahei .

generated 4- Labeled DNA fragments are separated bused on naviccur nN bv tupilirv gi

electrophoresis, and their ussociated fluorescence is detected by a Crurgeoagped vic
(CCD). 5- A chromatogram is generaled based on peak fiuer N:'ne'e dtei, ).g
o fluorophure-labeled dlNTPsinucorportedateachpostion o e
from Sanger Sequencing", by BioRenler.com (202
NGS is a
high-throughput method achieved by sequencing clonally ampliied DNA K.
on
a massively paralel scale. The speed, throughput, and accuracy achieved with N
revolutionized genetic science and enabled applications once thought science of as fiction
What is Next Generation DNA
Sequeneing?
n contrast to microarray methods, sequence-based approaches directly determine the nucleie
acidsequence of a given DNA or cDNA molecule.
The first
major foray into DNA sequencing was the Human Genome Project. This project,
which used
first-generation sequencing, known as Sanger sequencing (the chain-temination
method), took 13 years, $3 billion and
cost was completed in 2003.
Compared conventional Sanger sequencing using capiliary electrophoresis, the short
to
massively parallel sequencing technique is a fundamentally different read,
revolutionised sequencing capabilities and launched the approach that
methods or next-generation second-generation sequencing
sequencing (NGS) that provide orders of magnitude nmore data
-

at much lower
recurring cost.
Nexi-generation sequencing (NGS), also known as high-throughput sequencing, is the caich-
all term used to describe a
number of different modern
These technologies allow for sequencing technologies.
sequencing of DNA and RNA much more
quickly and
han the
previously used Sanger sequencing, and as such revolutionised the study of cheaply
and molecular
biology. Such technologies include: genomics
Advantages of NGS
NGS can be used to analyse DNA and RNA samples and is a popular tool in functional
genomics. In contrast to nmicroarray methods, NGS-based approaches have several advantages
including:
a
priori knowledge of the genome or genomic features is not required
it offerssingle-nucleotide resolution, making it possibie to detect related genes (or features).
altenatively spliced transcripts, allelic gene variants and single nucleotide polymorphisms
higher dynamic range of signal
requires less DNA/RNA as input (nanograms of materials are sufficient)

higher reproducibility
NGS technologies

Illumina (Solexa) sequencing: Illumina sequencing works by simultaneously identifying DNA

bases, as each base emiis a unique fluorescent signal, and adding them to a nucleic acid chain

Roche 454 sequencing: This method is based on pyrosequencing, a technique which detects
pyrophosphate release, using light signal (bioluminescence), atter nucleotides are incorporated
by polymerase to a new strand of DNA. Roche 454 sequencing platform has been discontinued
since 2016.

lon Torrent: Proton / PGM sequencing: lon Torrent scquencing measures the direct release of
H+ (protons) from tne incorporation of individual bases by DNA polymerase and therefore
differs from the previous two methods as it does not measure light.

2
 1972:Sanger started work on DNA sequencing
sequencing
9r.Sanger developed Oindeoxy thain terminaton method of DNAof DIA SEqUercug
1977.Maxan and Gitbert developed chemic al degradation method
First 1977 First ONA based gename sequenc ed (x 1/4 harteriophage
9 9 5 First bacterium Maemophllus infionzoo was sequenced by sholgun methoc
h
Generation 996 Applied Biosystems developed automated DNA sequencing b a s e d on S a r g e r s i

PPo Firsteukaryotic gencme (Saccharomyces cergvisiae) was sequenced

leams
pubished by two di fferent independent
2001.First huhan penome dralt was

2005 Fiust NGS platforin released Roche 454 GS-20

2006 Introduction of second NGS platform-Solexo Ger ome Analyzer
2006 nitiation of 1000 gencme project
454 GS-FLX 2 AB SOLD seqcencer
2007. Introduction ot Roche
2008. Developeent of lurina GA-I
Ttanicm
Second 2009 Introduction of Roche 454 GS-FI.X
2010 ntroduct.dn of Roche 454 G9.Junicr
Ilumlha 1Seq
Generation 2011 1ntrocucton ot SOLiD 5500W &
2012 Introduct:on of liunna HiSeq
8 lurlina uinSeq
2013. Introduction af SOLiD 65007| 5O0 &
Intrductin of Roche 454GS-Junuor+, ilurnina Ne1Seq
201
2017 inroduction of Ium.na iSeq 100
eicoe ,
Ceeloomet offrst commercial plaito.1n Gtthrd generation technckogy ie
2008.
Helicose Siascierces
f.lactime PG
reessod tte Persorial Genone
201C. Ion T:re7
2011 In:rocuctOnof PacEio RS C:C2
Third XL á a c 5 . c RS: C2 XL. Ion
Torrent releas
2 0 1 2 Introcuicn af PacBia RS C2
Generation 2013. Introduct n of PatEia RSI1
C2 XL
PS C3 & PacEic R3I PS C4
231 I:tocucticn cfPa:£isRSI1 520.
201S: introcuctionaf ior $5:55XL 53 540
2 3 1 6 1m ocueic of Pacfiz sequel

Te:hndoges
21 Re'ease I : l © N ala!fom by No G Nan3 201e
GridON & Sm agiON X3 olarfonis by Ortorc
2017 Reease ct FrolathON.
Fourth Mancpo3 lcg.es
p'altt'm by -sord Nanopore ecnrc ag e
Generation 213 CorTerc alza: cn ci PrckletT-©A

and introduction of ditferent generations' sequencing

Sequencing events, developments, order
platforms in chronological

Next Generation Sequencing

Technology based on three things

(A) Chemistry (B) Detection (C) Amplification

involved
Requred or all secod
Cptical tCCD, 2 )
Sequercing by Ligation generation sequencers

y Enuls:on 6 Bndge
Sequencing by S) n!hess Soid Stte uleclu0n
CR Amphicaton

Not requed ler thud and

Nanopore L Electncal'Detection çurth generaton sequencers

1
harpore

oeneratinn comenrin ( S) nn the hoaic ftmor fto

lassifiration of the next 1

dcicciivii
>y >Lcr d i i i
Covai dppiiuaiio
icSuy,

3
 End Repair 8
Fragmentaüon (Adaptve Adapter Ligation
SampeGencme Focused Acousbe Sheartng)

Clonal Amplificaton
by Bridge PCR
Cional Ampiificaticn
by Emusion PCR

DNA I s

G.,
L Lscferar

Pytosequencing Sequepcing ty igation Sequercing by synthesis

Roche 454)|

Schematic representation of the basic steps invoived in DNA sequencing using ditterent NGS

platforms

Introduction to NGS
Next-generation sequencing (NGS) is a technology for determining the sequence of DNA or
RNA too study genetic variation associated with diseases or other biologica!
phenomena. introduced for commerciai use in 2005, this method was initially calied
"massively-parallel sequencing", because it enabled the sequencing of many DNA strands at
the same time, instead of one at a time as with traditional Sanger sequencing by capillary
electrophoresis (CE).
Each of these technologies has utility in today's genetic analysis environment. Sanger
sequencing is best for analyzing small numbers of gene targets and samples and can be
accomplished in a single day. It is also considered the gold-standard sequencing technology.
so NGS results are often verified using Sanger sequencing. NGS enables the interrogation of
hundreds to thousands of genes at one time in multiple samnples, as well as discovery and
analysis of different types of genomic features in a single sequencing run, from single
nucleotide variants (SNVs), to copy number and structural variants, and even RNA fusions.
NGS pro, ides the ideal throughput per run, and studies can be performed quickly and cost-
effectively. Additional advantages of NGS include lower sampie input requireneuis, iigier
accuracy, and ability to detect variants at lower aliele frequencies than with Sanger sequencing

revolutionized genetic analysis and enabled

The speed, throughput, and accuracy of NGS has
new applications in genomic and clinical researeh. reproductive health. and environmental

agricultural, and forensic science.

Nent-Generation Sequencing workflow
ypical NGS experiment shares similar steps repardless of the instrument technology cd
Figure )

1 Construct 2.Clonal 3. Sequence 4.Analyze

Library Amplification Library Data

Figure 1. NGS worktlow steps

1. Construct library
is
sequencing "library must be created iîrom the sample. The DNA (or cDNA) sanple
the
processed into reiatively short double-stranded tragments (100-800 bp). Depending on

specific application. DNA fragmentation can be performed in a variety of ways, including

physicai shearing, enzyme digestion, and PCR-based ampilticati.on of specific genetic regions.
The resulting DNA fragments are then ligated to technology-specific adaptor sequences,
have a nique molecular "barcode", so
forming a fragment library. These adaptors may also
allows for multiple samples to
each sample can be tagged with a unique DNA sequence. This
For example, barcodes 1-20 can be used to
be mixed together and sequenced at the same time.
them in a single sequencing run. This approach,
individually label 20 samples and then analyze
or "multiplexing", saves
time and money during sequencing experiments and
called "pooling"
controls for workflow variation, as pooled samples are processed together.
two other specialized methods of library preparation:
In addition to fragment libraries, there are
Paired-end libraries allow users to sequence the
paired-end libraries and mate-pair libraries. which occurs only in a single
DNA fragment from both ends, instead of typical sequencing
libraries, but they have adaptor
direction. Paired-end libraries are created like regular fragment
from two directions. This
the DNA insert that enable sequencing
tags on both ends of detection of genomic
reads and can be used to improve
methodology makes it easier to map or splice
elements, and RNA gene fusions
rearrangements, repetitive sequence tools have
modem library prep methods and analysis
variants. However, improvements in well.
made it possible to detect these features with single direction sequencing as
lo create than pared-end libraries and
fragment or
Mate-pair libraries are more complex
of mate-pair
DNA inserts (over 2 kb and up to 30 kb). Sequencing
involve much larger-sized the opposite orientation. Using
that are distal to each other and in
libraries generates two reads sequencing
assoCiated between the two sequencing
reads, mate pair
the physical information identitication of complex
structural variant detection, and
is useful for de novo assembly, large
genomic rearrangemenls.

ronal m nlifingtinv

solid surface and clonally an1plified

DNA library must be attached to a
Prior to sequencing. the sequencing. During this
that can be detected trom each target during
io increase the signal
 bound to the
surface ol a bead or a fn
nmolecule in the library is Torrent technolog
Pess, cach unique
DNA
clones. In the
case of lon
Celt and PCR amplified to ereate a set of identical molecules to
beads.

"templatingis used to add library

process called
3. Sequence library
a sequencing
time using
at the same
the library is sequenced all utilize a version
of the
o r the DNA in is unique, they
NGS technology
nstrument. Although each individual bases as they grow
along a polymerized
Sequeneng by synthesis" method, reading on single
stranded DNA,
DNA base synthesis
steps:
T his is a cycle with common
removal of reactants to
Strand. base, and then subsequent
TOlowed by detection of the incorporated
restart the cycle.
nucleotide incorporation
detection to determine
Most sequencing instruments use optical detection to sense the
release of
lon Torrent instruments use electrical
during DNA synthesis. DNA
when nucleotides are incorporated during
hydrogen ions. which naturally
occurs

synthesis.
. Analyze data
of short DNA
of conmplex data consisting
Each NGS experiment generates large quantities and data anaiysis toois, they
has its own aigorithms
reads. Although each technology platform evaluate the quality of NGS
data
share a similar analysis 'pipeline
and use c o n m o n metrics to

seis.
nd tertiary analy sis (Figure
be divided into three steps: primary, secondary.
Anaiysis instrument detectors into digitized
can

is the processing of raw signals from

2). Primary analysis each sequencing cycle. The output
of
These data are collected during
data or base calls. raw
reads (FASTQ files)
files containing base calls assembled into sequencing
primary analysis is involves read
scores (Plued quality scorc).
Secondary analysis
and ther associated quality to a reference genome
on quality,
followed by alignment of reads
and trimming based isa
filtering variant calling. The main output
for novel genomes, and finally by
or assembiy of reads it invoives
reads. Tertiary analysis is the most challenging step. as
BAM fiie containing aligned information from the data.
results and extracting meaningful
interpreting

PrmaryAnalisis pucFASTa
TerergAalys
Antarerotaandtresul
ouaudnedorinab Qput;3A
Aited
reads andRUiy

overview
Figure 3. NGS analysis pipeline
is typically
NGS data and associated
algorithms, NGS analysis
the complexity of don't have specialized
Due to users who
bioinformatics specialists. To empower intuitive
performed by ike lor "Torrent have created user-friendly.
bioinformatics training. platforms shiiis tw gel iesuiis. ioi a

and doesn't require programning

software that simplifies analysis see the
article The Importance of

basic metrics used to analyze NGS data please

primer on the
Throughput and Coverage.

6
Overview of methods
(NGS) methods lo cno
have ultiple next-generation sequencing
uenomes researchers methods include wn
their studies. General NGS
rom when designing and implementing subdivided into exome
which is futher
and targeted sequencing. they can all yiela
Sc e Scquencing panels (see table below). Although While whole
o r region-specific
Sequencing and gene far from interchangeable.
interest to a researcher. they targeted next
are

ene sequences of discovery-based questions,

are suited to there are
whole-exome sequencing
Senome and each of these approaches
is preterred. Additionally. within diseases, or regions
of the
generation sequencing organisms,
tailored to specitic sample
types, includes examples
specalized techniques method and
and cons of cach
below summarizes the pros
enome. The table
and applications.
methods
DNA sequeneing
Comparison of
Targeted sequencing:
Targeted region-specif+c
Whole genome or
exome gene
sequencing:
scquencing (WGS) paneis
sequencing
Scquencing regions of
Sequencing only disease-
Sequencing entire gencme interest such as
Deseription exons (protein associated genes, or
coding regions)
genomic hotspots

Highly tlexible,
Most comprehensive
1% of human customizable
Pros genome, much
genome coverage
of less data to designs
Detect widest range Data is focused
anaiyze than
features: SNVs, specitically on
indels. structural and WGS regions/genes of
copy nunmber variants,
Faster
workilow than interest
regulatory elements
Lowest sample input
PCR WGS
No bias from (10 ng)
amplification or probe Multiplexing Compatible with
small number
hybridization FFPE tissue
of samples
Best for discovery Multiplexing large
Medium sample
research numbers of samples
input (50 ng-1
Better for detecting
ug depending
rare alleles
on library prep
method)

Only get data on

A lot of potentially Only get data in

Cons exons (may
targeted regions
unnecessary data from may miss relevant
iss
non-coding/non- variants if not in
functional regions functionally
relevant design)
Data is very
variants)
complicated
May be too
Multiplexing usually
much extra data
not possible
if one only
needs to study a

SIlaii nuiber

of genes
 Speed/ Slowest Medium Fastest
return of
results

Data
volume Largest Mediun Smallest

When to
use
Complete coverage of Disease- Clinical sequencing
genome needed specific
De oTO assembly Disease-specific
research research projects
scovery of projects
L.known genomic
IVD testing
Clinical LDT development
variants causing a
disease sequencing Inherited disease
Aneupioidy detectioon Oncology
preimplantation Immune repertoire
geneie testing) Liquid biopsy

ros and Cons

ge ijde
Targeted NGS
Fast and cost-effective ior Higher sequencing depth for
iow
target increased
number sensitivity (down to 1%)
Advantages Established workflow Higher discovery power
Simple data analysis Higher mutation resolution
Longer reads (500-700 More data
generated with the same
bps) DNA
quantity
|Low sensitivity Higher throughpui
(15-20% detection
limit) Less cost-effective for
low
Disadvantages Low discovery power
(1-20 targets)
target number
Low
scalability due to increasing Time-consuming for low target number
sample input requirements (1-20 targets)
Next-generation
scientists can ask sequencing
Short reads
a broad range of and answer. technology has
fiundamentally changed (150-300 bps) by Illumina
Innovative sample the kinds of
applications. For preparation
example, NGS ailows and data analysis questions
1.
Rapidly sequence whole labs lo. options enable
2.
3.
Deeply sequence genomes
target regions
Utilize RNA
or sequencing
quantify mRNAs for (RNA-Seq) to discover
4.
Analyze epigenetic factors
gene expression novel RNA
analysis variants and
such as splice sites
Sequence cancer genome-wide DNA
6.
Study the human sap'es to Stidy methylation and
7.
ldenify novel microbiome
rarc
soi.iatic v di idiiis,
iullioi
DNA-protein
pathogens SubCines, ana more
What is sequencing?
DNA
Sequencing is the process of determining the sequence of nucleotide bases (As,
Is, Cs, and Gs) in a piece of DNA. Today, with the right equipment and materials.
Sequencing a short piece of DNA is relatively straightforward.
Sequencing an entire genome (all of an organism's DNA) remains a complex Lask.
it requires breaking the DNA of the genome into many smaller pieces, sequencing
the pieces, and assembling the sequences into a single long "consensus." However.
new methods that have been developed over the past two decades, genome
sequencing is now much faster and less expensive than il was during the luman
Genome Project

SangerScquencing:
What s Sanger sequcncing2
Sanger sequcncng. also known as the "chain termination nnethod," was devetoped
by the Englisochcmist Frederick Sanger and his colleagues in 1977. This method
is based on amplitication ofthe IDNA fragment to be sequenced by DNA polymerase
and incorporation of modified nucleotides specifically, dideoxynucleotides
ddNTPs).
This method is designed for dctermining the sequence of nucleotide bases in a piece
of DNA (commonly less than 1.000 bp in length). Sanger sequencing with 99.99%
base accuracy is consicored the "gold standard" for validating DNA sequences,
including those already quenced through nexl-generalion sequencing (NGS).
Sanger sequencing was ised in the Human Genome Project to delermine the
sequences of relatively small fragments. of human DNA (900 bp or less). These
fragments were used assemble larger DNA fragments and, eventually, entire
chromosomes.

Ingredients for Sanger sequencing

Sanger sequencing involves making many copies of a target DNA region. Its
ingrediens are simiiar to those needed ior DNÁ repication in an organism, or lor
polymerase chain reaction (PCR), which copies DNA in vitro. They include:

(1) A DNA polymeraseenzyme

(2) A pimer, which is a shiort piece of single-slranded DNA ihat binds to thec
template DNA and acts as a "starter" for the polymerase
3) The tour DNA
nucleotides (dATP, dTP, dCTP, dGTP)
(4) The
template DNA to be sequenced
However, a
Sanger sequencing reaction also contains a unique ingredient:
(O) Dideoxy, or chain-terminating, versions of all four nucleotides
ddCTP, ddGTP), cach labeled with a (ddATP, ddlTP
different color ol dy

-oCH Base
H H

Dideoxynucleotide (ddNTP)

-OCHa Base
H

OH

Deoxynucleotide (dNTP)
Dideoxy nucleotides are similar to regular. or deoxy, nucleu.ides, but with one kcy
difference: they lack a hydroxyl group on the 3" carbon
of the sugar ring. In a regular
nucleotide, the 3' hydroxyl group acts as a "hook," allowing a new nucieotide to be
added to an existing chain.

Once a dideoxy nucleotide has been added to the chain, there is no

hydroxyl
available and no further nucleotides can be added. The chain ends with the
dideoxy
nuclcoide, which is marked with a particular color of dye depending on the base (A.
T, Cor G) that it carries.
 low Does Sanger
Sequencing Work?
Anger sequeneing, DNA
DNA be
to
a
primer conplementary to the template DNA (re
sequenced) is used to be a starting point for DNA
presence of the four synthesis. In tne
deoxynucleotide triphosphates (dNTPs: A, G, C, and 1), tne
polymerase extends the primer
by adding the complementary dNTP 1o the
DNA strand. To
delermine which nucleotide is template
nucleotides, four dideoxynucleotide incorporated into the chain of
ddCTP, and ddTTP) labeled with a distinct triphosphates (ddNTPs: ddATP, ddGTP
fuorescent dye are used to terminaie the
synthesis reaction. Compared to dNTPs, ddNTPs has an
the ribonucleotide. hence cannot
oxygen atom removed from
fornn a link with the next nucleotide.
synthesis, the reaction products are loaded into four lanes
Following
(on the diverse
of a single gel depending
chain-terminating nucleotide and subjected to gel electrophoresis.
According to their sizes. the sequence of the DNA is thus deterinined

SangerSequencing Steps
The Sanger sequencing method consists of 6 steps:
1) The double-stranded DNA (dsDNA) is denatured into two single-stranded DNA
(ssDNA).
(2) The DNA sample is divided into four separate sequencing reactions.
containing
ail four of the standard deoxynucleotides (dATP, dGiTP, dCTP and dTTP) and
the DNA polymerase. To each reaction is added only one of the four
dideoxynucleoides (ddATP, ddGTP, dCTP, or ddTP). The dúNTPs may be
radioactively or fluorescently labelled for detection in automated sequencing
machines.

(3) Primer that corresponds to one end of the sequence and poBymerase solution are
added to all four tubes.

(4) The DNA synthesis reaction initiates and the chain extends until a termination
nucleotide is randomly incorporated.

(5) he resulting DNA Irag1nents are denatured inlo sslDNA.

(6) The denatured fragments are separated by gel electrophoresis and the sequence
is determined. This is frequently performed using a denaturing polyacrylamide-
urca gel with each of the four reactions run in one of four individual lanes (lanes
A. T. G.C). The DNA bands may then be visualized by auloradiography or UV
light and the DNA sequence can be directly read off the X-ray film or gel image.
 Heat
-
AGTCATGAGTCC5 Template
5 3 Punor
1 2 3 4
5 -AGTCATGAGTCCS
3

TGTT
ATP
GTP dATP ATP
DNA
ol Tol
DNA ToTPrimer
DNA
dCTP
CTCP
dGTP
dCTP dGTP Fol
dAT
PolDNA Polymeras
OTCP dTTP dATP
dTCP dCTP drEE. dTTP Eree
TdATP SdGTP doTp dcy?,7deoxynucleotid
dG72 dGTP
ddATP
ddGTP
DNA polymarase ddCTPDideoxynucleot
For example

3 AGTCATGAGTCC
5 TCAddG
3 AGTCATGAGTCC-5

5*TCAGTACTCAcdG
5 AGTCATGAGTCC
TCAGTACTCAGddG

Complementary
chain

Electrophoresis

Figure: Sanger sequencing protocol

Autoradiography
In order to
identify various terminated chains of DNA (the DNA
to be
radioactively or fluoresccntly labelled beforehand. The DNAstrands)
strands
the ddNTPs would have
are then
gel electrophoresis; then. the gcl is read from
top to bottom (3" to 5) to obtain the
separated using
strands nigrale the bottom, whiie
io sequence Smaller
molecule in order to find the DNA
iarger. strands stay up 1op (near the well). We can read each
sequence

AUTOMATED DNA-SEQUENCING (Modification of Chain Terminatio

Method)
 It
uoaic DNA sequencing is bascd on S:mger method of DNA scquene"E
makes use of Automatic Sequencers
cti
containing raaroat
nuclcotides
uomatcd sequcneing include tagging with st the 5
end witn
d

labeled
lor radiolabelling, or using a primer system
Tor
Osphorus rcading in an optical
sequencing facilitates by
CScent dye. Dye-priner automation The
later development
analysis and
tStCr
more economical
and
labeled ddNTPs
and primers
set the
coworkers luorescently
of imethod
LCTO HoOd and Chain-termination

DNA sequcncing.
Stge automated. high-throughput
ior recently. dyc-terminator
scquencing

DNA sequencing. More ot the chain

navegreatly simplilicdDye-terminator sequencing utilizes labellingratheT than tou
nas been developed. reaction.
in a single
which permits sequencing
sequencing.
cach o
erminatorddNTPs. method. In dye-terminator
rcactions as in the labelled-primer labelled with
fhuorescent dy es. eac

chain terminators is
the tour dideoxynucleotide
waveiengtns.
iwhich emit ight at different
of

10 iG
20 TC CGC..G 1GC
THOCCGCCG
OCCcTTTIC r TG ca
C1G

ol a Sanger sequencing
This is an example ot the output DNA bases are
Chromatograph: The tour
Figure: labclled dyc-icrminators. the
fluorescently the sottware to give
read using which are interpreted by
different colours
represented by
DNA sequence above.
up to 584
can sequence
instruments (DNA sequencers)
Automated DNA-sequencing DNA sequencers cary
a day.
(run) in up to 24
runs

DNA samples in a single

batch
detection and recording of
for size separation,
out capillary electrophoresis fluorescent peak trace chromatograms.
and output as
data
dye fluorescence entire genomes.
Aulonation has lead to
the sequencing of

STEPS:
poly1nerase chain reaction(PCR)
. Labelling the products of
4. In place ofradioactive labelling, luorescent lables are usedl. The luorescent lables
are attached to the four dideoxyribonucleotides or dedNTPs (ddATP, ddGTP, ddC IP
and ddlTP) used for chain
termination.
. lf
ditferent fhuorochrome is attached to cach of the four ddNTPs, all of them
a

may be used in the same reaction (the

Single Track System).
4. In a
single track system, the reaction products are run in a single lane of gel or
capillary
. The
reaction products are
subjected to
polyacry lamide gel clectrophoresis (PAGE)
under denaturing eonditions.
6. The bands produced in polyacry lamide gei are identificd with the help of
tluorescent detector, which identifies the fluorescent
signal emitted by each band.
7. The fluorochromes are
excited by laser bean and the
sensed
resuluing
fiuorescent signai
is
by a photovoltaic ceil.
8. The resulting data are fed into a
computer. which converts these signals into the
base sequence of the DNA molecule

9. The sequence information (data) can be

for future use
printed out or stored in data storage device