COURSE # MBFS 518: TRENDS IN NUCLEIC
ACID SEQUENCING TECHNIQUES
NEW METHODS OF SEQUENCING
Dr. MUHAMMAD ASIF NAEEM
ASSOCIATE PROFESSOR
Maxam–Gilbert sequencing
Maxam–Gilbert sequencing is a method of DNA
sequencing developed by Allan Maxam and Walter
Gilbert
in
1976–1977.
This
method
is
based
on nucleobase-specific partial chemical modification of
DNA and subsequent cleavage of the DNA backbone at
sites adjacent to the modified nucleotides.
Maxam–Gilbert sequencing was the first widely adopted method
for DNA sequencing, and, along with the Sanger dideoxy
method, represents the first generation of DNA sequencing
methods. Maxam–Gilbert sequencing is no longer in widespread
use,
having
been
supplanted
by
next-generation
sequencing methods.
Allan Maxam and Walter Gilbert’s 1977 paper “A new method for
sequencing DNA” was honored by a Citation for Chemical
Breakthrough Award from the Division of History of Chemistry of
the American Chemical Society for 2017. It was presented to the
Department of Molecular & Cellular Biology, Harvard University
Procedure
Maxam–Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA
fragment to be sequenced (typically by a kinase reaction using gamma-32P ATP)
and purification of the DNA. Chemical treatment generates breaks at a small
proportion of one or two of the four nucleotide bases in each of four reactions (G,
A+G, C, C+T). For example, the purines (A+G) are depurinated using formic acid,
the guanines (and to some extent the adenines) are methylated by dimethyl sulfate,
and the pyrimidines (C+T) are hydrolysed using hydrazine. The addition of salt
(sodium chloride) to the hydrazine reaction inhibits the reaction of thymine for the Conly reaction. The modified DNAs may then be cleaved by hot piperidine; (CH2)5NH
at the position of the modified base. The concentration of the modifying chemicals is
controlled to introduce on average one modification per DNA molecule. Thus a
series of labeled fragments is generated, from the radiolabeled end to the first "cut"
site in each molecule.
The fragments in the four reactions are electrophoresed side by side in
denaturing acrylamide gels for size separation. To visualize the fragments, the gel is
exposed to X-ray film for autoradiography, yielding a series of dark bands each
showing the location of identical radiolabeled DNA molecules. From presence and
absence of certain fragments the sequence may be inferred.
Single Base Chain Extension Technique
Introduction to Single Base Chain Extension (SBCE)
Single Base Chain Extension (SBCE) is a molecular
biology technique used to extend a primer by a single
nucleotide. This method is primarily employed in
applications such as genotyping, DNA sequencing, and
the analysis of genetic variations. SBCE is particularly
useful for identifying single nucleotide polymorphisms
(SNPs), which are variations at a single nucleotide
position in the DNA sequence among individuals.
Principle of SBCE
The principle of SBCE involves the use of a primer that is
complementary to a target DNA sequence, except for the
last base, which is designed to be extended by a single
nucleotide. DNA polymerase is then used to extend the
primer by incorporating a single nucleotide that is
complementary to the base on the template strand. The
reaction is typically carried out in the presence of dideoxy
nucleotides
(ddNTPs)
or
other
chain-terminating
nucleotides to ensure that the extension is limited to a
single base.
Applications of SBCE
•Genotyping: SBCE can be used to determine the genotype
of an individual at a specific locus by identifying the nucleotide
incorporated during the extension reaction.
•DNA Sequencing: Although next-generation sequencing
(NGS) technologies have become prevalent, SBCE can still
be applied in certain sequencing applications, especially
when high accuracy is required for specific bases.
•Genetic Variation Analysis: SBCE is useful for analyzing
genetic variations, including SNPs, insertions, and deletions,
by extending primers across regions of interest.
FIRST-GENERATION SEQUENCING
Maxam-Gilbert Method
Sanger Sequencing: Old Method
Sanger Sequencing: New Method
SECOND-GENERATION SEQUENCING
Roche 454 Method or Pyrosequencing
Illumina acquired the Solexa Genome Analyzer
The Solid method, the ligation sequencing-based platform
Ion Torrent Method
THIRD GENERATION SEQUENCING
PacBio sequencing method
The Oxford nanopore technology method
Second-Generation DNA Sequencing
Second-generation sequencing technology allows
for the rapid sequencing of the whole genome. For
this NGS, there are several different kits and
equipment possibilities. These tools and kits are
designed to make the procedures cost-effective
and time-saving. As second-generation novel
sequencing techniques, It Makes use of the Roche
454, Illumina, SOLİD, and Ion Torrent platforms.
Roche 454 Method
The Roche/454 GLS FLX Titanium instrument uses a platform
known as pyrosequencing. Emulsion PCR and the subsequent
pyrophosphate detection technique are the foundations of
Roche/454 pyrosequencing.
Nyrén et al introduced the pyrosequencing concept in 1997. It is
a continuing procedure that combines magnetic beads covered
with streptavidin, 3'-deficient recombinant DNA polymerase, and
luciferase. 5’ exonuclease activity (read-out evidence) and
luminescence detection bring the process to a close.
Illumina Method
Illumina's Illumina Method is a sequencing system that uses
reversible dyes to create polonies through bridging PCR. It
involves joining adaptor segments to single strands and
amplification. Four types of nucleotides are introduced, each
fluorescently labeled with a different color. Unincorporated
nucleotides are washed away, followed by laser elimination
to eliminate the fluorescent probe and blocking group. The
cycle begins when one base develops a distinct fluorescent
color, enabling sequence identification.
Solid Method
Applied Biosystems purchased a ligation sequencing-based
platform in 2006, which has a 99.85% accuracy rating for
data collection. It uses the Roche 454 emulsion PCR
method, which uses magnetic beads to affix DNA fragments
to a glass flow cell plate. DNA ligase sequences amplified
fragments using two-base-coded probes, with four distinct
colors representing each base pair. The probe with the
correct base pair is added to the sequence, and the ligation
cycle restarts when the fluorescent dye is removed.
Ion Torrent Method
This method involves shifting a primary base back in each cycle
to complete the sequence. After five cycles, there is enough
information to fill in gaps. This method is suitable for small RNA
and gene expression analyses, whole-genome sequencing, and
targeted region sequencing. The technique uses optics-based
techniques to gather photons from genome sequences and
information on base pairs. Fragmented DNA is joined to
microbeads in a microchip with flow cells and electrical sensors.
Protons are found using a semiconductor sensor, and the pH of
the solution changes as hydrogen ions are released.
Third-Generation DNA Sequencing
With the increasing use of DNA sequencing methods,
new technological developments are needed. Although
first- and second-generation sequencing methods are
revolutionary for DNA sequencing, they need to be
improved in many aspects, such as time, cost, and
error rate. As a result of these searches, thirdgeneration sequencing methods with longer read
lengths, low cost, and faster sequencing have been
developed
Pacific Bioscience Method
Pacific Biosciences' PacBio method is a single-molecule realtime (SMRT)-based method used to read long-dimensional
DNA sequences. It inserts hairpin-shaped sequences called
"SMRTbell" at the beginning and end of a double-stranded
DNA sample, minimizing base errors. The DNA binds to
polymerases immobilized on a zero mode waveguides (ZMW)
chip, allowing for real-time analysis. The method uses
fluorescently labeled dNTP to detect epigenetic modifications
and structural changes in the DNA sequence. This method is
still in development and is the most commonly used platform
among third-generation sequencing methods.
Oxford Nanopore Technology Method
This is a third-generation DNA sequencing method
developed by Oxford Nanopore Technologies. It uses an
artificial nanopore embedded in the membrane with an
electric current, formed by alpha-hemolysin (αHL). The
DNA is introduced into the nanopore, recognized, and
transported
by
a
motor
protein.
The
algorithm
characterizes the molecule passing through the nanopore
based on the interruption of the ionic current. ONT can
read in a short time.
Future Approaches
Sequencing techniques are utilized in diagnosing and
treating complex diseases like cancer, offering new insights
into disease diagnosis and treatment. These methods
identify unknown genetic variants, examine DNA, RNA, or
protein
levels,
detect
mutations
in
cancer
signaling
pathways, and narrow individual genetic profiles. Comparing
transcriptome readings between patients and healthy
individuals helps identify early disease diagnosis and
personalized treatment methods.