DNA SEQUENCING

(Assignment)
Submitted to: Dr. Hafiz Abdullah Shakir
Submitted by: Nabia Fiaz (MS Roll no. 02)
Fiza Adil (MS Roll no. 04)
Muqadas Sardar (MS Roll no. 05)
Subject: Advanced Analytical techniques
Session: 2022-2024
Table of contents:
DNA sequencing
 Purpose of DNA sequencing
General steps in all DNA sequencing techniques
1. Reactions
 Base specific reactions
 Enzymatic extensions
2. Separation
 Slab gel sequencing
 CE sequencing
3. Detection
 Radioactive
 Fluorescence
DNA sequencing methods
 Basic DNA sequencing
o Maxam-Gilbert sequencing
o Sanger sequencing
 Next generation DNA sequencing
o Hybridization
o Pyrosequencing
o Nanopore sequencing

Maxam-Gilbert sequencing:
o Procedure
o Advantages
o Disadvantages

Sanger sequencing
o History
o Principle
o Material
o Methodology
o Advantages
o Disadvantages
o Automation of sequencing
Next generation sequencing:
1. Hybridization:
o Definition and explanation
o Principle
o Working
o Applications
2. Pyrosequencing
o Definition
o Steps of pyrosequencing
o 454 Approach steps
3. Nanopore sequencing:
o Introduction
o Theory of nanopore sequencing
o Types of nanopore sequencing
 Biological membrane systems
 Solid state sensor technology
o Applications
 Disadvantages of next generation sequencing
 References
 DNA Sequencing
DNA Sequencing:
DNA Sequencing is the technique that determines the exact order of the four nucleotides
bases such as adenine, thymine, cytosine, and guanine that make up the DNA molecule (Mardis,
2017).
DNA double helix contains four bases called nitrogenous bases bond each other through
base pairs. Adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). Th
human genome has 3 billion copies of these base pairs. These base pairs contain all the
information to maintain human body. So, these base pairs one can easily determine the processes
of transcription, translation and also used for sequencing of DNA molecules, providing basics to
sequencing methods (Mardis, 2017).
Purpose:
Data obtained from DNA sequences has become essential for research purposes and in
many fields of science such as:
 Medical diagnosis
 Biotechnology
 Forensic biology
 Virology
 Biological systematics
Scientists can utilize sequenced data to find all the genes and regulatory systems present
in DNA molecule. DNA sequencing also seen its application in medicine field like diagnosis and
treatment of diseases. DNA Sequencing also has the ability to reform food quality and safety. It
also performs its function in maintaining sustainable agriculture for plant, animal, and public
health b by animal breeding and protecting from disease outbreaks (Mardis, 2017).
The rapid speed of sequencing attained with modern DNA sequencing technology has
been instrumental in the sequencing of complete DNA, or genomes of numerous types and
species of life, including the human genome and other complete DNA sequences of many animal,
plant and microbial species. DNA sequencing techniques are playing key roles in many applied
fields and many science fields are taking advantages of these techniques like archaeology,
anthropology, genetics, biotechnology, molecular biology, forensic sciences, many more.
DNA sequencing is also used in many new discoveries to promote revolutionizing (França,
Carrilho, & Kist, 2002).
 Biotecnology
Genetics

DNA Forensic
Sequencing science

Medical Molecular
diagnosis Biology

Figure 1: Applications of DNA sequencing in other applied fields

General Steps in all DNA sequencing techniques:

All types of DNA sequencing techniques consist of basically a reaction, a separation, and
detection and data analysis. The sequencing reactions are base-specific reactions and enzymatic
extensions utilizing DNA polymerases. Separation involves polyacrylamide gel electrophoresis
(PAGE) or capillary electrophoresis (CE). The most common detection methodologies include
fluorescence, although radioactivity has been used previously. Each of these steps is discussed in
more detail (Nunnally & Brain, 2005).
1. REACTIONS:
The sequencing reactions can involve base-specific reactions or enzymatic extensions
utilizing DNA polymerases.
a) Base-specific reactions:
The Maxam–Gilbert method utilize base-specific chemical degradation reactions to regulate
the sequence of radioactively 5’ end-labeled DNA fragment. It is appropriate for both single- and
double-stranded DNA and need no DNA polymerases. Four samples of radioactively end-labeled
fragments are base-specifically chemically cleaved and separated electrophoretically in four
separated lanes based on the specific reactions employed.
b) Enzymatic extensions:
The Sanger Method uses enzymatic extensions utilizing DNA polymerase to produce
partially synthesize DNA fragments by terminating the replicating chain of DNA fragment. In
this double stranded DNA fragment is denatured to produce single stranded template strand
which needs to be sequenced. Then this ss-DNA template is mixed with DNA primer,
polymerase enzyme, and four nucleotide molecules (dNTPs) into four tubes. The buffer solution
is also added to maintain the pH.
A special ddNTP is also incorporated in each tube. This special ddNTP produce partially
synthesize fragments of DNA by terminating reaction. The sequence of the template DNA is
determined by DNA sequencing (i.e., PAGE) gel and then gel is exposed to x-ray film to develop
bands. The sequence is read from bottom to top. The complementary strand of visualized strand
will give the original template sequence (Nunnally & Brain, 2005).
Base-specific reactions utilizing
Chemicals
1. Reaction Enzymatic extensions utilizing DNA
polymerases

Polyacrylamide gel electrophoresis

2. Separation (PAGE)
Capillary electrophoresis (CE)

Detection methodologies include

radioautography using
3. Detection Fluorescence
Radioactivity

Figure 2: General Steps in all techniques of DNA sequencing

2. Separation:
The separation of DNA fragments is done by electrophoresis. The electrophoresis is a
process use to separate molecules i.e nucleic acids and proteins on the bases of charge and size
by using specific gel.
a) SLAB-GEL Sequencing:
The novel DNA sequencing techniques used standard slab PAGE instrument for separation
and visualization of the end products of the DNA sequencing reactions. The only PAGE gel
electrophoresis is not actual experiment. Electrophoresis is totally the separation of molecules
having different charges but all DNA molecules have same negative charge. These DNA
fragments are separated on the basis of size or length of fragments of varying lengths.
The polyacrylamide gel is used which creates an excess of “pores” having different sizes.
DNA molecules having large sizes become tangled in these pores. The smaller fragments move
quickly through these pores and move towards bottom. The larger fragments appear at the top of
gel machine. The fragments are obtained in four different lanes. Radioactively labeled DNA
fragments are used. The composition of these gels is mostly 6% acrylamide in 1 × TBE (tris-
borate-EDTA) buffer.
b) CE Sequencing
The advance DNA sequencing technique is Capillary Electrophoresis sequencing. This
sequencing system was developed after sab-gel sequencing.
 The CE system permitted for increased speed, easy to use, and better accuracy. Sequence of all
DNA fragments is obtained in signal lane. This system also uses fluorescent dyes incorporated in
DNA fragments instead of radioactively labelled fragments.
CE separations offer several advantages over slab-gel-based sequencing systems. First,
capillary systems have dynamic coatings and can be used several times. Secondly, there is no
need to synthesize gel which must be poured, as gels are difficult to synthesize without bubbling
also time consuming. Thirdly, the stretchy capillaries are easily attached to a microtiter plate.
Finally, multi-capillary systems create significantly increase the throughput of a sequencing
system (Nunnally & Brain, 2005).

Figure 3: DNA Sequencing by Using Capillary electrophoresis method

3. Detection:
a) Radioactive:
Previously, detection was achieved by radioactive labels of different elements such as 32P or
35
S. Radioactive labels as tags were tremendously effective and beneficial for detection of DNA
sequencing for reaction products. The labeled reagents such as primer or nucleotide are not
dissimilar in length or size or shape than the unlabeled one, so the DNA polymerases show no
preference or any kind of fidelity reductions.
However, radioactive elements are very dangerous to deal with them and radioactive gels must
be directed to the x-ray film. This step takes at least 24 to 36 h to create the bands and to collect
500 bases of sequencing DNA (Maxam & Gilbert, 1977).
 b) Fluorescence:
The fluorescence sequencing system was first developed in Hood’s laboratory about in mid-
1980s. The system used different dyes; all dyes showed different emission maximum.
 Fluorescein isothiocyanate
 NBD-amino-hexanoic acid
 Tetramethyl-rhodamine isothiocyanate
 Texas Red
The use of fluorescence-based systems has removed radioactive labels from almost all types
of DNA sequencing. The reason behind this is increased safety, easily disposable, multiplex
ability and acquiring of real-time data. The most important parameter of all is multiplex ability of
these fluorescent dyes.
Only one well with four dyes is used instead of four lanes as in PAGE gel. Eventually, real-
time is obtained without using X-ray film and off-line data collection (Rosenthal et al., 1990).
DNA Sequencing Methods:
Basically, there are three main types of DNA sequencing:
1. Basic DNA sequencing
2. Advanced DNA sequencing
3. Next Generation DNA sequencing

Basic DNA Sequencing Method

1. Maxam-Gilbert DNA sequencing method.
2. Sanger chain termination DNA sequencing method

Next Generation DNA sequencing

1. Pyrosequencing
2. Nanopartical DNA sequncing
3. Hybridization DNA sequencing

Figure 4: Pictorial diagram Showing DNA sequencing techniques

Basic DNA Sequencing Methods:

Basic DNA sequencing methods were given by scientist in 1970-1977. These are classical
Methods of DNA sequencing. It includes:
1- Maxam- Gilbert Method of DNA sequencing
(Chemical Degradation Method of DNA Sequencing)
2- Sangar DNA sequencing method
(Chain termination method of DNA Sequencing)
1. Maxam-Gilbert Method of DNA sequencing:
The conventional DNA sequencing method was given by Allan Maxam and Gilbert and
published in 1977, therefore named as Maxam-Gilbert chemical cleavage method of DNA
sequencing. This method also called Chemical degradation method. The method requires no
DNA polymerase. This is an initial step to sequence a DNA molecule So, this method describes
the first generation of DNA sequencing methods (Maxam & Gilbert, 1977).
Principle:
This method uses chemicals for chemical modification of nitrogenous bases present in
particular DNA fragment and cleavage of these modified bases to produced DNA fragments of
different lengths. Different chemical reagents are used for this purpose. These fragments of
different lengths are separated by gel electrophoresis into four separate lanes. Then, sequence is
read out by using radioautograph on X-ray film and this sequence is used for particular work of
our interest (Verma, Kulshrestha, & Puri, 2017).

1. Preparation of 2. Modification of
3. Cleavage of 4. Electrophoresis
radioactively end nitorgenous bases
DNA fragment at and reading of
labeled ss-DNA of that particular
modified bases. sequence
Fragement. DNA fragement

Figure 5: Principle of Maxam-Gilbert Method of DNA sequencing

Procedure:
This method uses double stranded DNA molecule as a starting material which needs to be
subsequent changes. The overall procedure requires the following steps:
1) Preparation of radioactively 5’-end labelled ss-DNA
2) Modification of nitrogenous bases
3) Cleavage at modified base position
4) Separation of cleavage DNA fragments by Electrophoresis plus autoradiography
 5) Analysis/ Reading of DNA sequence
1) Preparation of radioactively 5’-end labeled ss-DNA:
This method requires radioactively labelling of 5’-end of DNA with a radioactive element i.e
32
P. For this purpose, multiple reactions are performed on double stranded DNA molecule.
 Firstly, dephosphorylation of ds-DNA is done to remove naturally incorporated P at
5’end. This reaction is favored in presence of Alkaline phosphatase enzyme.
 Secondly, radioactive element ¿- 32P)-dATP are incorporated with 5’end of both DNA
strand (from which the normal P were removed). This reaction is occurred in the presence
of enzyme polynucleotide kinase taken from E.coli (Verma, Kulshrestha, & Puri, 2017).

Phosphorylation of ds-DNA with ( -

Dephosphorylation 32
P)-dATP
of ds-DNA
(Polynucleotide kinase)
(Akaline phosphatase)

Gel Electrophoresis ( Separation &

Denaturation of ds-DNA
isolation)
(Dimethyl sulphoxide & 90 C)
Radioactively labled ss-DNA

Figure 6: Preparation of radioactively labeled DNA strand

 Now the two strands of DNA are separated by using dimethyl sulphoxide and then heated
at 90 C.
 These strands are further subjected to gel electrophoresis for separation and isolation. The
separation is based on heavy and lighter strands as purines (Guanine & adenine) are
heavier than pyrimidines (Thymine & cytosine).
 The single strand of DNA labelled with γ - 32P is divided into separate samples ready to
treat with chemical reagents.
 Figure 7: Synthesis of radioactively labeled single stranded DNA at 5' end γ - 32P

2) Modification of nitrogenous bases:

This method is basically chemical treatment of radioactively single stranded DNA fragment
to be sequenced. The set of single strands of DNA labelled with γ - 32P are taken into four
different test tubes.
Then these tubes are treated with chemicals cause modification of bases and cleavage of
glycosidic bond present between deoxyribose sugar and base (Verma, Kulshrestha, & Puri,
2017).

Table 1: Different Chemicals used for Cleavage of Different Bases

Base Specificity Tube 1: Tube 2: Tube 3: Tube 4:

G base G+A Bases C+T Bases C Base
i. Chemical used for Dimethyl- DMS + Formic Hydrazine Hydrazine +
Base alteration sulphate acid Alkali (NaCl)
DMS
ii. Chemical used for Piperidine Acid Piperidine Piperidine
altered base removal (Formic acid)
iii. Chemical used for Piperidine Piperidine Piperidine Piperidine
Strand cleavage
 a) Tube 1→G base:
After adding DNA fragments, Dimethylsulphate DMS is added in this tube. The DMS
chemical cause methylation of G bases at N (7) and hydrolysis of glycosidic bond. This removes
the G base as a depurinated residue.
b) Tube 2 → A+G bases:
In this tube, only DMS is not enough as it methylates A at N-3 position rather than N-7. So,
formic acid is added which removes the both A and G bases at comparable rates from DNA
fragment.
c) Tube 3 → C+T bases:
This tube is treated with hydrazine (NH2-NH2) which breaks the glycosidic bond and remove
both cytosine and Thymine.
d) Tube 4 → C base:
If DNA fragment is treated with hydrazine (NH 2-NH2) in 1.5M NaCl, then only C will
remove from the fragment. It will help to compare C and C+T cleavage positions help in
identification of T residues (Verma, Kulshrestha, & Puri, 2017).

1. Gylcosidic Bond 2. Phosphodiester bond

Chemical treatment Cleavage of DNA
Removal of base
to break Fragment
(DMS, Hydrazine, Acid) (Piperidine)

Figure 8: Chemical treatment to break different bonds in DNA molecule

3) Cleavage at modified base position:

After modification and removal of bases from DNA fragments, piperidine is added in all four
tubes. The hot piperidine chemical breaks the phosphodiester bond present between two
nucleotides at the points where bases are missing. It is highly specific chemical breaks DNA
fragment only from one position. Thus, at the end of this treatment a set of nested set of end-
labelled DNA fragments of different lengths are produced.
 Figure 9: Base-specific alteration and cleavage to produce DNA fragments of different
lengths

4) Separation of cleavage DNA fragments by Electrophoresis plus autoradiography:

After chemical treatment, the cleavage fragments are separated on their size basis using
polyacrylamide gel electrophoresis. The cleavage samples of all four tubes are run on a
sequencing gel. The sequencing gel separates these fragments depending on their size. Fragments
are visualized as bands by autoradiography. The cleavage fragments of samples of DNA like, G,
G+A, C+T and C are simultaneously run at a temperature of 70C in four vertical lanes in
electrophoresis. The sequencing gel is a long thin 0.1× up to 20cm polyacrylamide slab. This
slab contains approximately 8M urea (Verma, Kulshrestha, & Puri, 2017).

Figure 10: Separation of DNA fragments and reading of sequenced DNA using
polyacrylamide gel electrophoresis
 5) Analysis/ Reading of DNA sequence:
The DNA sequence is read directly from the gel. First, the bands are visualized by using X-
ray film autoradiography.
Advantages of Maxam-Gilbert Method:
Advantages of Maxam and Gilbert method of DNA sequencing are as follows;
 It is used to sequenced both single and double stranded DNA molecule.
 DNA replication is not required so no need of DNA polymerases and dNTPs. There is
also no need of premature termination of DNA template. So, no problem with polymerase
to synthesize DNA.
 Stretches of DNA fragments can be sequenced which is not possible with enzymatic
method.
 Sequenced and purified DNA can be read directly without complementary the sequenced.
 Homo polymeric DNA runs are sequenced as efficiently as heterogenous DNA
sequences.
 It is also used to study foot-printing i.e DNA interactions with proteins.
 Can be used to analyze the nucleic acid structure and epigenetic modifications the DNA.
 Figure 11: Schematic diagram showing Maxam-Gilbert Chemical Degradation Method of DNA
sequencing

Disadvantages of Maxam-Gilbert Method:

Disadvantages are as follows;
 Modern techniques like automated DNA sequencing and next generation DNA
sequencing eradicate this method so currently is not widely used.
 This method uses radioactivity and toxic chemicals, hazardous for public health and
safety
 It needs extensive amounts of expensive and hazardous chemicals.
 It cannot sequence more than 500 base pairs.
 Not easy to scale up and have relatively complex set-up.
 The read length decreases from incomplete cleavage reaction.
 It is difficult to make Maxam and Gilbert sequencing kits (Jagadeeswaran & Kaul, 1986).
Sangers Sequencing
History
Chain termination sequencing and dideoxy sequencing are other names for this method. The
genomic landscape of the human genome could not have been mapped out without the help of
Sanger sequencing. It was invented in 1975 by Frederick Sanger, but it did not enter commercial
use until 1977 (Sanger, Nicklen, & Coulson, 1977). Sanger sequencing method of DNA
Sequencing was first commercialized by Applied Biosystems. It was the most widely used
sequencing method for approximately 40 years (Shendure & Ji, 2008).
Principle
Dideoxy ribonucleoside triphosphates, which are lacking a 30-hydroxyl group, are the primary
building blocks of the approach (Verma, Kulshrestha, & Puri, 2017). Sequencing by synthesis,
also known as Sanger dideoxy DNA sequencing, creates a complimentary copy of a single-
stranded DNA template using a DNA-dependent polymerase (SBS). DNA polymerases will
incorporate a chain-terminating 2′,3′- dideoxynucleotide triphosphate (ddNTP) at the appropriate
complementary position, but synthesis will be terminated by the incorporation of the ddNTPs at
the 3′ ends because the next nucleotide to be added lacks the required 3′ hydroxyl group for
dNTP phosphodiester bond formation (Sanger, Nicklen, & Coulson, 1977; Valencia et al., 2013).

Figure 122: dNTP (with a free 3' -OH) is added to the DNA strand that is being synthesized
during synthesis. However, strand synthesis halts when a ddNTP is inserted because there is
no 3' -OH to create a phosphodiester bond with the subsequent dNTP (Verma, Kulshrestha, &
Puri, 2017)

Material
There are multiple individual parts to this method that all work together to carry out the
sequencing.
 ssDNA template to be sequenced.
 Primers.
 Taq Polymerase for template strand amplification.
 Buffer.
 Deoxynucleotides (dNTPs).
  Fluorescently labeled dideoxynucleotides (ddNTPs) (Verma, Kulshrestha, & Puri, 2017).
Primer designing: Primers can be designed in several ways, either manually or with the use of
software (Primer3, PrimerBLAST, etc.). Primer development requires attention to several
specific factors.
1. Primers need to be between 15 and 28 bases in length.
2. The recommended ratio of base constituents is 50-60% (G+C).
3. Primers should terminate (30) in a G or C, CG or GC; this eliminates end "breathing" and
improves priming efficiency.
4. Temperatures between 55 to 80 degrees Celsius are ideal (even 80 degrees is too high,
however many of the gaps were discovered to be GC-rich).
5. Primer's 3' ends shouldn't form base pairs, as this would lead to the preferred synthesis of
primer dimers rather than the intended result.
6. It is preferable to use primers that do not have self-complementarity (the ability to
produce secondary structures like hairpins) (Verma, Kulshrestha, & Puri, 2017).
Methodology
 Recently extracted DNA can undergo direct sequencing without any additional processing. In
PCR, the denaturation, annealing, and extension steps can be repeated for 25-30 cycles.
 The dsDNA is denatured, or broken apart, into two ssDNA molecules (ssDNA).
 To obtain a continuous succession of synthesis products that reflect each, possible chain
termination location, four reactions involving template, polymerase, all four dNTPs (one
radioactively labeled), and primer are set up.
 An attached primer spans the gap between two consecutive bases in the sequence.
 A polymerase is added to a mixture that already contains dNTPs of four different types but only
one of the ddNTPs.
 Additionally, one of the four ddNTPs is present in each reaction, with the amount present
representing the likelihood of incorporation.
 Due to the absence of a 3' OH group in the integrated ddNTP, a phosphodiester bond cannot be
formed between the C3' OH of the sugar moiety and the C5' of the subsequent dNTPs, causing
the chain to break.
 In each of the four reactions, several strands have been terminated, and their lengths range
widely.
 Due to the presence of a single ddNTP species in each reaction, various fragments of varying
lengths are produced, with each fragment ending at a place in the template sequence
corresponding to one of the four nucleotides.
 Single-nucleotide resolution is achieved by separating the four reactions separately on a large
denaturing polyacrylamide gel. After denaturing the DNA in each of the four reaction mixes,
the DNA is separated into single strands by electrophoresis in parallel lanes on a high-
resolution polyacrylamide gel.
 The core sequence of the analyzed template can be read directly from the band pattern
throughout the four lanes. Find the first band at the bottom of the sequence to begin reading it.
Continue on to the subsequent longer section, and so on (Sanger, Nicklen, & Coulson, 1977;
Valencia et al., 2013; Bajpai, 2014 Verma, Kulshrestha, & Puri, 2017).

Figure 13: The Sanger sequencing method in 6 steps (adapted from Gauthier, 2008).

Advantages
The following are the characteristic pros of this method:
 High single-pass accuracy.
 Good ability to call repeats.
 Long read lengths.
 Relatively simple workflows and data analysis.
 Easily accessible software. (Verma, Kulshrestha, & Puri, 2017).
Disadvantages
The following are the cons of this method:
 Low throughput.
 High cost of Sanger sample preparation.
 Time-consuming.
 Not suitable for sequencing large genomes.
 High infrastructure cost. (Verma, Kulshrestha, & Puri, 2017).
Automation of sequencing
Sanger sequencing was first introduced, and further changes to the process for automating
sequencing were made. Applied BioSystems (now part of Life Technologies) developed the first
generation of "automated sequencers" in 1986, which featured automation of the gel
electrophoresis stages, detection of the fluorescent DNA band patterns, as well as analysis of
bands, marking the end of the period of manual sequencing. The use of dye-terminator
sequencing is an example of such progress. The primary benefit of this technology is the
increased precision and swiftness it provides. A machine designed by Leroy Hood and Mike
 Hunkapiller and released by Applied Biosystems, Inc. (ABI) in 1987, the ABI model 370 was the
first automated DNA sequencing machine and could produce read lengths of up to 350 bp per
lane. In 1995, ABI also released the ABI PRISM 310 Genetic Analyzer, which was designed to
make pouring gels, installing the instrument, and loading samples easier and more streamlined.
The capillary sequencer was invented by Swerdlow and Gesteland; it uses polyacrylamide gel-
filled capillaries rather than slab gels. There are sequencers on the market right now that can
accommodate 4, 16, 48, 96, or 384 capillaries. The read length and sequencing speed both
improved as the number of capillaries expanded (França, Carrilho, & Kist, 2002).
Modern automated DNA sequencing takes use of capillary electrophoresis, which analyzes 8-96
sequencing operations simultaneously, and the newest generation of fluorescent dyes, which
produce strong and unique fluorescent emissions. Key advantages of "first automated DNA
sequencing" implementations over methods described for the original Sanger sequencing
included the removal of radioactivity use, "one-lane" sequencing, "one-tube" reactions,
automated base calling, and the replacement of slab gel technology with multi-capillary
electrophoresis with automatic, electrokinetic-injection lane loading (Valencia et al., 2013).

Figure 14: Processing protocols of Maxam-Gilbert and Sangers Sequencing. (Verma, Kulshrestha, &
Puri, 2017).

Next generation DNA sequencing methods:

Hybridization:
Definition:
The process in which two complementary single-stranded DNA and/or RNA molecules bind
together to form a double-stranded molecule is called hybridization. The bonding is based on the
complementary base-pairing across the two single-stranded molecules. Hybridization is a vital
process in many research and clinical laboratory techniques.
Explanation:
The hybridization method of sequencing DNA was developed in the 1980’s. A DNA sample can
be sequenced after cosmid DNA has been inserted into M13 vector. There are many markers for
identification of unique DNA inserts on the plate, but the plate can also have contaminants.

Figure 15: Cosmid DNA Figure 16: M13 vector

For the recognition of unwanted sequences chemiluminescent hybridization has been
developed. Labelled DNA is used to investigate and determine the usefulness of useless
sequences. For this objective, an antibody is bind with the label DNA and then it is exposed to
certain chemicals. The antibody emits light as a reaction. If a series of 96-well plate is probed
then it is possible to sequence only those samples that are with yeast insert.
Principle:
The principle of hybridization is the binding of single stranded DNA/ RNA with complementary
strands. This widespread method uses two probes. “Probes are DNA oligonucleotides (15-1000
nucleotides long) of known sequences which can be radioactively or fluorescently labelled”.
These oligonucleotide sequences are bind with the DNA to be sequenced. The non-required
DNA sequence is removed by the process of continuous hybridization, whether the hybridizing
labelled fragments matched the sequence of the DNA probes on the sieve.
Working:
M-mer Probe:
A probe is called an m-mer probe when it becomes attached with DNA and becomes positively
expressed. It becomes the substring of DNA sample. The length of oligonucleotide is usually
denoted by –mer, it is Greek word that means parts. The combination of positively expressed
probes is known as spectrum of DNA samples.
Washing out of unwanted sequences:
If one is using a 4-mer probe for a single-strand of DNA sample with the sequence 5′GGTCTCG
3′, only five probes will hybridize with the single strand DNA.

Figure 17: Hybridization of DNA with probes (Zhang et al., 2011)

According to Drmanac et al., (2002) all other probes will form mismatch hybrids at the end base
and during selective washing they will be denatured. The five probes that are of good match at
the end base will result in fully matched hybrids, which will be kept and detected. In another
report by Drmanac et al., Format 1 synthesis by hybridization, a large number of DNA samples
are attached onto a nylon membrane, which is utilized as a solid support to form the DNA array.
The probes in hybridization:
The first probe is with the DNA from which the library was created, as it removes out the
substance and the second probe is the vector DNA from which the source DNA was created but
without accidentally picked inserts. The second insert is for example cosmid without insert. It
screens out M13 with largely useless sequences.
The first probe will thus remove any material that has no sonicated DNA while the
second probe removes that DNA which has no yeast sequences. A third probe can also be
introduced in this protocol to screen out the overlapping regions in DNA.

General applications:
 It was thus feasible to prepare larger contiguous sequence information, based upon
overlapping sequences from the probe hybridization spots. Sequencing by hybridization has been
largely relegated to technologies that depend upon using specific probes to interrogate
sequences, such as in diagnostic applications for identifying disease related single nucleotide
polymorphisms (SNPs) in specific genes or identifying gross chromosome abnormalities.
Detection of mutations:
The spectrum of DNA samples which has subset of probes is complementary to the
reference sequence is recovered from a stock of all possible probes of a given length.
Hybridization of probes with the control and patient samples then takes place. In the event where
a mutation is present, there is a mismatch between the probe and the DNA sequence; hence, the
probes do not bind to the test sample. The overlapping of probes results in a low percentage of
positively expressed probes at the mutation sites, indicating that the test sample sequence differs
from the control DNA.

Pyrosequencing:
Definition:
Pyrosequencing is a method of DNA sequencing based on the "sequencing by synthesis"
principle, in which the sequencing is performed by detecting the nucleotide incorporated by a
DNA polymerase.
It was the first next generation technique to gain commercial introduction in 2004. This
method sequences short stretches of DNA. In pyrosequencing, whenever a nucleotide of DNA is
inserted by DNA polymerase, a pyrophosphate is released. This pyrophosphate initiates a series
of downstream reactions that at the end produces light by firefly enzyme luciferase. The more the
nucleotides inserted the more light is produced (up-to the point detector can detect it).
Steps of pyrosequencing:
1. Single stranded DNA is hybridized to a sequencing primer, PCR amplifies this
hybridized DNA. Then, it is incubated with DNA polymerase, ATP sulfurylase,
luciferase and apyrase enzymes, while substrates which are utilized in this technique are
adenosine 5’-phosphosulfate and luciferin.
2. One of the four deoxy-nucleotide tri-phosphate (dNTP) is added to initiate the next step.
DNA polymerase incorporates dNTPs into the template DNA if it is complementary. The
amount of dNTPs added equal to the release of pyrophosphates (PPi), if the incorporation
occurred at all.
3. The enzyme ATP sulfurylase converts PPi into ATP with the help of substrate adenosine
5’-phosphosulfate. This ATP starts converting luciferin into oxy-luciferin that produces
visible light. The visible light is proportional to quantity of ATP. A charged coupled
device (CCD) camera detects the visible light produced in luciferin mediated reaction and
 a computer program can analyze this detected amount of light. Thus, the more the
number of nucleotides incorporated, the more the light signals.
4. The degradation of nucleotides is essential for continuing the sequences process. To
accomplish it apyrase was added in the first step, it removes all dNTPs from the solution.
5. Now if more nucleotides are added it can initiate a new cycle.
This sequencing technique is advanced for whole genome sequencing and it is considered one of
the fastest sequencing methods (Bajpai, 2014).

Figure 18: Steps of pyrosequencing

454 pyrosequencing approach steps:
In the Roche/454 approach, following steps are performed:
1. Library preparation: DNA samples is converted into small fragments of 300-800bp by
spray method, and adds different adapters at both ends. Otherwise, use primers for
amplification after DNA denaturation, clone into specific vectors, and finally making
single stranded DNA library.

Figure 19: Library perparation

2. The library fragments are mixed with a population of agarose beads whose surfaces carry
oligonucleotides complementary to the 454-specific adapter sequences on the fragment
library, so each bead is associated with a single fragment.
3. Each of these fragment: bead complexes is isolated into individual oil: water micelles that
also contain PCR reactants, and thermal cycling (emulsion PCR) of the micelles produces
approximately one million copies of each DNA fragment on the surface of each bead.

Figure 20: Emulsion PCR

4. These amplified single molecules are then sequenced altogether. First the beads are
arrayed into a pico-liter plate (PTP; a fused silica capillary structure) that holds a single
bead in each of several hundred thousand single wells, which provides a fixed location at
which each sequencing reaction can be observed.
 Figure 21: Sequencing
5. Enzyme containing beads that catalyze the downstream pyrosequencing reaction steps are
then added to the PTP and the mixture is centrifuged to surround the agarose beads. On
instrument, the PTP acts as a flow cell into which each pure nucleotide solution is
introduced in a stepwise fashion, with an imaging step after each nucleotide incorporation
step. The PTP is seated opposite a CCD camera that records the light emitted at each
bead.
6. The first four nucleotides (TCGA) on the adapter fragment adjacent to the sequencing
primer added in library construction correspond to the sequential flow of nucleotides into
the flow cell. This strategy allows the 454 base-calling software to calibrate the light
emitted by a single nucleotide incorporation. However, the calibrated base calling cannot
properly interpret long stretches of the same nucleotide (homo-polymer run).

Nanopore sequencing:
Nano pore is named so because it is a small hole of diameter of nanometers. Certain
cellular proteins penetrate across the membrane are called transmembrane proteins that act as
nanopore. Nanopores are also cut as a larger holes (tens of nanometer wide) in a silicon piece
and then the hole is gradually filled by use of electron beam methods and consequently small
holes (nanopore) is left.

Figure 22: Nanopore sequencing

Theory of Nanopore sequencing:
The theory of nanopore sequencing is based on the phenomenon that occurs when this nanopore
is dipped in a conducting solution and a voltage potential is applied across it. Under such
conditions a slight current develops due to movement of ions through nanopore. The amount of
current that flows through this nanopore is related to the size of nanopore very much.
The electrophoresis will take place if DNA is present in the solution. The DNA molecules start
their movement towards the nanopore and will pass through it as well. However, the small size
of nanopore ensures that DNA passes through the hole under the force as a long strip, one base at
a time passes through it. Each nucleotide of DNA obstructs the nanopore to a different degree
and this degree of obstruction is characteristic of each nucleotide.
The voltage across the nanopore is very sensitive to the size of the nanopore, it specifically
varies for each passing nucleotide i.e. the potential for A, T, C or G is characteristic. As a result
as one reads the change in current with the passage of DNA molecule it represents a direct
reading of DNA sequence. This way a single molecule of DNA can be sequenced by passing it
through Nano pores, PCR and other hybridization processes become useless.
Types of Nanopore sequencing:
There are two types of nanopore systems for DNA sequencing:
 Biological membrane systems
 Solid state sensor technology
Biological membrane systems:
Biological nanopore sequencing depends on the use of transmembrane proteins interspersed in a
lipid membrane to produce the holes. There are two proteins that have been used to generate
pores have been extensively studied:
1. Alpha hemolysin
2. Mycobacterium smegmatis porin A (MspA).
The rate at which DNA passes through the pores is maintained by the addition of motor proteins,
such as a highly processive DNA polymerase (phi29) that ratchets DNA through upon nucleotide
addition. DNA can be passed through the pores at a constant rate for tens of thousands of
nucleotides.
 Figure 23: Biological membrane system
Solid state sensor technology:
Solid state sensor technology utilizes many types of metals or metal alloy substrates with
nanometer sized pores that allow DNA or RNA to pass through.
In this nano pore sequencing technique proposed by McNally, the target DNA is first
subjected to a biochemical preparation step in which each base of the sequence is converted into
the form that can easily be read using a solid-state nano pore. In fact, each of the four bases in
the target DNA is converted to a predefined sequence of oligonucleotides, which is hybridized
with a molecular beacon that carries a specific fluorophore.
Molecular beacons are oligonucleotide probes that can report the presence of specific
nucleic acids in homogenous solutions. Molecular beacons are hairpin shaped molecules with an
internally quenched fluorophore whose fluorescence is restored when they bind to a target
nucleic acid. For a two colors readout (that is two types of fluorophores), the four sequences are
combinations of two predefined unique sequences, bit ‘’0’’ and bit ‘’1’’, such that an A would be
‘’1 1’’, a G would be ‘’1 0’’, a T would be ‘’0 1’’ and finally a C would be ‘’0 0’’. Two types of
molecular beacons carrying two types of fluorophores hybridize specifically to the ‘’0’’ and ‘’1’’
sequences.
The converted DNA and hybridized molecular beacons are electrophoretically passed
through a solid-state pore where the beacons are sequentially removed. Each time a beacon is
removed, a new fluorophore is lighted, which results into a burst of photons recorded at the
location of the pore. This method allows wide field imaging and specially fixed pores that make
possible the simultaneous detection of several pores using a special camera, electron multiplying
charged coupled device (EM-CCD) (McNally et al., 2010; Clark et al., 2009; Tyagi et al., 2000).

Figure 24: Solid state Nanopore sequencing

Applications:
Nanopore technology has been used to sequence environmental and metagenomic
samples, is currently on the space station, and has been used for bacterial strain identification.
Nanopore technology is also able to identify base modifications, similar to PacBio technology,
enabling epigenetic events to be readily identified. Nanopore sequencing also offers direct RNA
sequencing, as well as PCR-free cDNA sequencing. So, it offers low cost DNA/ RNA
sequencing.

Disadvantages of NGS techniques:

The common problem with all current NGS technologies is the short length of reads (sequenced
fragments) and higher error rates than those of traditional Sanger sequencing method.
The short reads that are produced in these methods are especially problematic when large scale
DNA fragments, e.g., a whole genome is to be sequenced. This drawback is an issue especially
in sequencing new genomes and in sequencing highly rearranged genome segments such as one
might discover in cancer genomes or in regions of structural variation.
The quality of each base is also lower than that of Sanger chemistry reads. The quality scores
compress a variety of types of information about the quality of base calls into a readily usable
probability of error value (Metzker, 2010).
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