DNA Sequencing and

its types
A T G C……
What is DNA
Sequencing ?
The process of determining the order of nucleotides adenine (A),
thymine (T), cytosine (C), and guanine (G) along a DNA strand.

We need to know the order of nucleotide bases in a strand of

DNA for sequencing.

All the information required for the growth and development of

an organism is encoded in the DNA of its genome.

So, DNA sequencing is fundamental to genome analysis

and understanding the biological processes in general.
Classical Methods
1. Maxam and Gilbert Sequencing method
2. Sanger Sequencing method

Advanced Methods
3. Hierarchal shot-gun Sequencing method
4. Whole genome Shot-gun Sequencing method

Next Generation Sequencing (NGS)

5. Pyrosequencing
6. Sequencing by Synthesis (Illumina)
7. Roche 454
8. Nanopore sequencing
9. Single Molecule Real time Sequencing (SMRT)
Classical Methods
 Classical Methods

To determine the order of the nucleotide bases adenine,

guanine, cytosine, and thymine in a molecule of DNA two
methods
were used
1. Sanger; Chain Termination Sequencing method
2. Maxam and Gilbert; Chemical Sequencing method
These two methods are most popular conventional methods
Robotics and automated sequencing are based on
these methods
Advanced Methods
Shot-gun Sequencing method
1. Hierarchal Shot-gun Sequencing
2. Whole-Genome Shot-gun Sequencing
Next-generation
sequencing
 NEXT GENERATION SEQUENCING (NGS)

Several competing methods of Next Generation Sequencing

have been developed by different companies.

1. Pyrosequencing
2. Sequencing by Synthesis (Illumina)
3. Roche 454
4. Nanopore sequencing
5. Single Molecule Real time Sequencing (SMRT)
 1. Pyrosequencing
Pyrosequencing is based on the' principle where a
complementary strand is synthesized in the presence of
polymerase enzyme.

 In contrast to using dideoxynucleotides to terminate

chain
amplification in Sanger sequencing),
(as instead pyrosequencing the release
detects
nucleotides are added to of pyrophosphate
the DNA when
chain.
It initially uses the emulsion PCR technique to construct
the colonies required for sequencing and removes the
complementary strand.

Next, a ssDNA sequencing primer hybridizes to the end of

the strand (primer-binding region), then the four different
dNTPs are then sequentially made to flow in and out of the
wells over the colonies.

When the correct dNTP is enzymatically incorporated into

the strand, it causes release of pyrophosphate
In the presence of ATP sulfurylase and adenosine, the
pyrophosphate is converted into ATP.

This ATP molecule is used for luciferase-catalysed conversion

of luciferin to oxyluciferin, which produces light that can be
detected with a camera.

The relative intensity of light is proportional to the amount

of base added (i.e. a peak of twice the intensity indicates two
identical bases have been added in succession).
 2. Sequencing by Synthesis (Illumina)
1. The first step in this sequencing technique is to break up
the DNA into more manageable fragments of around 200 to
600 base pairs.

2.Short sequences of DNA called adaptors?, are attached to the

DNA fragments.

3. The DNA fragments attached to adaptors are then made single

stranded. This is done by incubating the fragments with sodium
hydroxide.

4. Once prepared, the DNA fragments are washed across the

flowcell. The complementary DNA binds to primers? on the
surface of the flowcell and DNA that doesn’t attach is washed
away.
5. The DNA attached to the flowcell is then replicated to form
small clusters of DNA with the same sequence. When
sequenced, each cluster of DNA molecules will emit a signal that
is strong enough to be detected by a camera.

6. Unlabelled nucleotide bases and DNA polymerase? are then

added to lengthen and join the strands of DNA attached to the
flowcell. This creates ‘bridges’ of double-stranded DNA between
the primers on the flowcell surface.

7. The double-stranded DNA is then broken down into single-

stranded DNA using heat, leaving several million dense clusters
of identical DNA sequences.

8. Primers and fluorescently-labelled terminators (terminators

are a version of nucleotide base – A, C, G or T - that stop DNA
synthesis) are added to the flowcell.
9. The primer attaches to the DNA being sequenced.

10. The DNA polymerase then binds to the primer and adds the
first fluorescently-labelled terminator to the new DNA strand.
Once a base has been added no more bases can be added to the
strand of DNA until the terminator base is cut from the DNA.

11. Lasers are passed over the flowcell to activate the

fluorescent label on the nucleotide base. This fluorescence is
detected by a camera and recorded on a computer. Each of the
terminator bases (A, C, G and T) give off a different colour.

12. The fluorescently-labelled terminator group is then removed

from the first base and the next fluorescently-labelled
terminator base can be added alongside. And so the process
continues until millions of clusters have been sequenced.
13. The DNA sequence is analysed base-by-base during
Illumina sequencing, making it a highly accurate
method. The sequence generated can then be aligned
to a reference sequence, this looks for matches or
changes in the sequenced DNA.
Steps involved in Illumina
3. Roche 454
4. Nanopore sequencing
 INTRODUCTION
 The fourth-generation DNA sequencing technology
 Studies the interaction between DNA and protein, as well
as between protein and protein.
 Have the potential to quickly and reliably sequence the
entire human genome for less than $1000, and possibly for
even less than $100.
 The detection principle is based on monitoring the
ionic current passing through the nanopore as a
voltage is applied across the membrane. When the
nanopore is of molecular dimensions, passage of
molecules (e.g., DNA) cause interruptions of the
current level, leading to a signal.
In the 1990s, Church et al. And
Deamer and Akeson separately
proposed that it is possible to
sequence DNA using nanopore
sensors
TYPES OF NANOPORES
Nanopore technologies can be broadly divided
into two categories:
1. Biological nanopore
2. Solid-state nanopore

More recently, hybrid nanopores have been

proposed to take advantage of the features
of both biological and solid-state
nanopores.
 5. Single Molecule Real time
Sequencing (SMRT)

Zero-mode waveguides allow real-time observation f

single molecule at high concentration. They are
photonic nanostructures that optically observed
single fluorescent molecule.