Nanopore sequencing is a fourth generation DNA sequencing technique that involves monitoring changes in electric current as DNA molecules pass through nanopores. There are two main types of nanopores: biological nanopores made of protein complexes like alpha-hemolysin, and solid state nanopores made in thin silicon nitride membranes. Nanopore sequencing has advantages of being label-free, producing long reads at high throughput with low material requirements, but challenges include slowing DNA translocation and reducing noise. Potential applications are in single molecule sensing for analysis of biomolecules.

1. BY: LEELESH SINGH

2. NANOPORE
SEQUENCING

3. OUTLINE
INTRODUCTION
TYPES OF NANOPORES
1. Biological nanopores
2. Solid-state nanopores
HOW NANOPORE WORKS?
 ADVANTAGES
 CHALLENGES
 APPLICATIONS
 CONCLUSION

6. 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.

7. Conti...
 Since DNA bases (adenine, thymine, cytosine and
guanine) are different from each other in atomic scale, it is
essential to collect base-specific information at atomic level
to correspond the DNA sequence with the measured signals.
According to the different types of the signals, the detection
methods can be roughly classified into two categories:
the electrical detection methods and the optical readout
method.

8. In the 1990s, Church et al. And
Deamer and Akeson separately
proposed that it is possible to
sequence DNA using nanopore
sensors

9. 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.
TYPES OF NANOPORES

10. Biological nanopores
 also called transmembrane protein
channels,
 are usually inserted into a substrate, such
as planar lipid bilayers, liposomes, or other
polymer films.
 Nanopores formed by pore-forming
proteins
 Examples: alpha-hemolysin,
MspA,
phi 29

11. ALPHA-HEMOLYSIN
 a-Hemolysin (a-HL, also called a-
toxin)
 a-HL is an exotoxin secreted by the
bacterium Staphylococcus aureus, a
human pathogen.
 This mushroom-shaped heptameric
protein normally forms pores in the
membranes.
 The limited pore size (1.4 nm) has
restricted its application in the
analysis of ssDNA, RNA, or small
molecules.

12. Mycobacterium smegmatis porin A
(MspA)
 Powerful nanopore for reading
information from four
nucleotides simultaneously.
 by Butler et al. in 2008.
 The channel of the MspA
octamer is 1 nm in diameter at
the minimal point, which is
relatively small and narrow,
compared to that of alpha-HL.
Thus, it can improve the spatial
resolution of ssDNA
sequencing.

15. The advantages of biological nanopores
include:
1.Their well-defined and highly-reproducible
nanopore size and structure.
2.Biological nanopores can be modified easily with
modern molecular biology techniques, such as
mutating the nucleotide sequence to change the
amino acid residue at a specific site.

16.  Disadvantages of biological
nanopores:
1. The evironmental demands( like temperature ,
electrolyte concentration and Ph) of nanopores to
keep there biological activities.
2. The fragility of lipid bilayer makes the biological
nanopore breakdown easily

17. SOLID STATE NANOPORE
 In 2001, Li et al.
 are made in silicon compound membranes
(Silicon nitride)
 More recently, the use of graphene as a
material for solid-state nanopore sensing
has been explored.

21. The portable MinION –– the first
handheld nanopore DNA sequencer

23. The significant
advantages of nanopore seq.
 Label-free,
 Ultra-long
 Reads (104–106 bases),
 High throughput, and
 Low material requirement.
 The nanopore approach is one option for the
fourth-generation low-cost and rapid DNA
sequencing technology.

24. Challenges:
 To slow down DNA translocation from
microseconds per base to milli seconds.
 To reduce stochastic motion of the DNA molecule
in transit in order to decrease the signal/noise
ratio

25. APPLICATIONS
 Nanopores as single-
molecule sensing
technologies have
great potential
applications in many
areas, such as
analysis of ions,
DNA, RNA,
peptides, proteins,
drugs, polymers, and
macromolecules.