DNA Sequencing

Chemical Degradation Method

(Maxam and Gilbert’s Method)

• Developed by Allan Maxam and Walter Gilbert in 1977

• Involves the base-specific chemical cleavage of an end-
labeled DNA segment to generate a nested set of
labeled molecules
• The partially cleaved DNA fragment is subjected to five
separate chemical reactions, each of which is specific for
a particular base or type of base
• The resulting fragments terminate at that specific base
followed by high-resolution denaturing gel
electrophoresis and detection of the labeled fragments
by autoradiography
Steps involved in chemical degradation method
•Restriction digestion and radiolabeling
•Base-specific cleavage reactions
•Polyacrylamide gel electrophoresis and autoradiography
• Restriction digestion and radiolabeling

ds DNA is digested with a restriction enzyme

↓
ds DNA fragment is radiolabeled at 5’-end with α-32P
• Base-specific cleavage reactions
Base-specific cleavage reactions are carefully designed to modify
on an average only one of the target bases in each DNA
Radiolabelled DNA fragments
↓
Strands separation by heating the sample at 90°C; Purification by PAGE; Divided
into 4 parts
↓
Base-specific modification by reagents that modify a specific
base(s) (see table)
↓
Treatment with hot piperidine (volatile secondary amine; 1 M in
water, 90°C) (this cleaves the S-P backbone at the site of modification)
↓
A set of end-labeled molecules ranging from one to several hundred nucleotides
yielded
↓
Removal of piperidine either by ethanol (EtOH) precipitation or vacuum drying or
a combination of both
• Polyacrylamide gel electrophoresis and
autoradiography

End-labeled DNA molecules (obtained after modification and cleavage)

↓
Resuspended in a buffer containing 90% formamide
↓
Denatured by heating to 90°C
↓
Separated by sequencing PAGE
↓
Subjected to autoradiography
↓
Read autoradiogram to determine the DNA sequence
 Chain Termination Method
(Sanger’s Dideoxy Method)

• Developed by Frederick Sanger in 1977

• The most widely accepted and used method for sequencing
DNA
• This method involves controlled synthesis of DNA to
generate fragments terminating at specific points by using
ddNTPs (dideoxyribonucleoside triphosphates)
– The replacement of dNTPs with 2’, 3’-ddNTPs in the DNA chain
terminates DNA synthesis
– ddNTPs are nucleotide analogs that lack the 3’-OH group that is
necessary for phosphodiester bond formation and chain elongation
• Steps involved:

Denature ds DNA
↓
Divide into 4 sets
↓

Anneal specific primer to ss DNA in 4 tubes

↓
Extend by DNA polymerase using 4 dNTPs (any one radiolabelled) and ddNTPs
(any one of the four in different tubes)
↓
Base-specific chain termination due to incorporation of ddNTPs in newly
synthesized strand
↓

Run sequencing PAGE to discriminate ss DNA chains differing in length by a

single nucleotide
↓
Autoradiography
↓

Read autoradiogram
• Four different reactions in separate tubes are carried out, each having
four dNTPs, small amount of single type of ddNTP, primer, and DNA
polymerase
• Thus, four sets of reactions have different type of ddNTPs (i.e.,
ddATP/d dCTP/ ddGTP/ ddTTP)
• In this reaction, either one dNTP is α-32P labeled
• The DNA polymerase reaction cannot discriminate between dNTP and
ddNTP and hence ddNTP can be incorporated in the growing chain
• These ddNTPs thus replace dNTPs at random positions during
template-directed synthesis of a DNA strand by DNA polymerase and
terminate DNA synthesis in a base-specific manner (as these lack 3’-
OH residues)
• After a suitable incubation period, DNA of each reaction mixture is
denatured, electrophoresed on a sequencing gel, and subjected to
autoradiography
 Automated DNA Sequencing
• Today virtually all DNA sequencing uses automated
sequencers
• The principle is the same as the Sanger’s method, but
the method of detection is different
• Either the primer or ddNTPs are fluorescently labelled
• Thus rather than running the gel for a finite time and
reading the result, the machine uses a laser to read the
fluorescence of the dye as the bands pass a fixed point
• Much longer sequences can be read from each track in
this way
• A further advantage is that the sequence read by the
machine is fed automatically into the computer
• Sequences ~700 bases in one reaction
• This is not only quicker than reading a gel manually and
typing the resulting sequence into a computer, but also
avoids the errors that are virtually inescapable with
manual data entry
• For large-scale genome sequencing projects, the
processes of sample preparation, setting up the
sequencing reaction, and loading the products into the
sequencer, are also automated, using robotic methods
• Two types of fluorescence labeling systems
– Four reaction/one gel system
– One reaction/one gel system
• Four reaction/one gel system
– The fluorescent primers can be used with nonlabelled
ddNTPs
– The primers used in each of four chain extension
reactions are 5’–linked to different fluorescence dyes
– The resulting fluorescent-labeled DNA strands are
separated in four different lanes in the electrophoresis
system
– This is the principle of the Amersham Pharmacia
Biotech ALF sequencer
– It has fixed argon laser that emits light, which passes
through the width of the gel and is sensed by
detectors in each of the lanes
• One reaction/one gel system
– Each of the four ddNTPs is labeled with spectrally
different fluorophore
– The chain extension reaction is carried out in a single
tube
– The resulting fragment is subjected to gel
electrophoresis in a single lane
– The tag is incorporated into the DNA molecule by DNA
polymerase and completes two processes in one step,
i.e., termination of DNA chain synthesis and attachment
of fluorophore at the end of DNA molecule
– This is the principle on which the original Applied
Biosystems Instruments (ABI) operate
– The gel is illuminated with an argon beam, and the
fluorescent signals are amplified and detected by
photomultiplier tubes [or a charge-coupled device (CCD)
camera]
– Since fluorescent signals can be detected and processed
in real-time, sequencing gels can be run for longer times
and more data can be collected by this technique
– Computer software (Base calling software) identifies
each nucleotide based on the unique color (emission
wavelength) of each dye to identify each base according
to the shape of the fluorescent peak and the distance
between successive peaks
– Because of their higher throughput, better consistency,
and lower sensitivity to electrophoretic artifacts, the
single lane instruments have gradually become the
approach of choice
Printout of automated sequencer (One reaction/one gel)
 Pyrosequencing
• More rapid ‘minisequencing’ method because it does not
require electrophoresis or any other fragment separation
procedure
• This method determines which of the four bases is
incorporated at each step in the copying of DNA
template
• In this method, dNTPs are used and ddNTPs are not
required
• As the new strand is being made, the order in which the
dNTPs are incorporated is detected, so the sequence
can be read as the reaction proceeds
• In the reaction, all four dNTPs (dCTP, dTTP, dGTP, α-thio
dATP) are not added at one time; rather each dNTP is
added individually in a sequential manner
• If the particular dNTP is not incorporated into the growing
chain, then it is rapidly degraded by nucleotidase or washed
before the addition of next dNTP
• The addition of a nucleotide to the end of the growing chain
is detectable because it is accompanied by the release of
pyrophosphate, which is detected in an enzyme cascade
that emits light
• The released pyrophosphate converts adenosine
phosphosulfate (APS) into ATP in the presence of enzyme
ATP sulfurylase
• Finally, on addition of luciferin and enzyme luciferase, ATP
is degraded into AMP that is accompanied with a flash of
chemiluminescence
Reactions involved:
DNA polymerase
(dNMP)n + dNTP → (dNMP)n+1 + PPi
• Since both ATP and dATP are used by luciferase, dATP
cannot be used for nucleotide incorporation; otherwise
false results may be obtained. Instead α-thio dATP, an
analog is used in place of dATP, which is used as a
substrate by DNA polymerase but not by luciferase
• All the four dNTPs are not added at one time because in
such a case, flashes of light would be seen all the times,
and no useful sequence information would be obtained
(any of the four will be added)
Gene chip array–based DNA sequencing

• DNA chips are used for simultaneously detecting and

identifying many short DNA fragments by DNA : DNA
hybridizations
• Also known as DNA array or oligonucleotide detector
that can be used for large-scale sequencing
For sequencing small test DNA:
Denature test DNA Glass chip (square
(to be sequenced) array 1 cm2)
↓ containing up to a
ss DNA million nucleotide
↓ probe sequences
Fluorescently labelled (as 8 base ss sequences)
↓
Dip glass chip in test DNA solution
↓
Test DNA hybridizes simultaneously to all possible stretches of 8 bases on
the chip
↓
Analyze using computer software
• For example,
– The sequence of test sequence is GCTA TGCT GGCT TACG
– Its complementary sequence will therefore be
CGATACGACCGA ATGC
– If this test sequence hybridizes to DNA chip having all possible
8-base sequences, the base sequences that will hybridize are
CGATACGA, GATACGAC, ATACGACC, TACGACCG,
ACGACCGA, CGACCGAA, GACCGAAT, ACCGAATG, and
CCGAATGC
• Steps involved in sequencing of larger DNA:

Cleave test DNA into smaller pieces

↓
Denature
↓
Tag with fluorescent dye
↓
Hybridize with ss probe DNA on chip
↓
Scan the chip by laser to locate the fluorescently-tagged DNA
↓
Analyze hybridization with the help of an attached computer
↓
Arrange the sequences into correct overlapping order by computer
software
• Before about 1975, nucleic acid sequencing techniques
lagged far behind those of protein sequencing, largely
because there were no available endonucleases that
were specific for sequences greater than a nucleotide
• Rather, RNAs were cleaved into relatively short
fragments by partial digestion with some RNases
– Ribonuclease T1 (from Aspergillus oryzae), which
cleaves RNA after guanine residues or
– Pancreatic ribonuclease A, which cleaves RNA after
pyrimidine residues
• Moreover, there is no reliable polynucleotide reaction
analogous to the Edman degradation for proteins
• Consequently, the polynucleotide fragments were
subjected to partial digestion with either of two
exonucleases:
– Snake venom phosphodiesterase, which removes
residues from the 3’ end of polynucleotides or
– Spleen phosphodiesterase, which removes residues
from the 5’ end
• The resulting oligonucleotide fragments were
identified from their chromatographic and
electrophoretic mobilities
• Sequencing RNA in this manner is a lengthy and
painstaking procedure
• The first biologically significant nucleic acid to be
sequenced was that of yeast alanine tRNA
• The sequencing of this 76-nt molecule by Robert Holley,
a labor of 7 years, was completed in 1965
• This was followed, at an accelerating pace, by the
sequencing of numerous species of tRNAs and the 5S
rRNAs from several organisms
• Entire 3,569-nt RNA genome of the bacteriophage MS2
was sequenced by Walter Fiers
• In contrast, DNA sequencing was in a far more primitive
state because of the lack of available DNA
endonucleases with any sequence specificity
• Present day technique:
– RNA can be easily sequenced by modifying DNA
sequencing techniques

RNA
↓ Reverse transcription
cDNA
↓
Sequence (as done for DNA)