The document discusses nanopores for DNA sequencing. It begins by defining nanopores as small holes typically 100 nm or smaller that can be used to identify molecules passing through. It then discusses several methods for DNA sequencing using nanopores, including using the alpha-hemolysin protein nanopore and MspA protein nanopore to detect individual nucleotides. Graphene nanopores are also discussed as a promising new method to discriminate between different nucleotide base pairs through their interaction with the graphene. The document highlights the potential for nanopore sequencing to be direct, fast and inexpensive compared to other sequencing methods.
Nanopore sequencing is a unique, scalable technology that enables direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore. The resulting signal is decoded to provide the specific DNA or RNA sequence.
Oxford Nanopore was founded in Oxford Nanolabs by Dr.Gordon Sanghera, Dr.Spike Willcocks and Professor Hagan Bayley. Nanopore sequencing has been around since the 1990s, when Church et al. and Deamer and Akeson separately proposed that it is possible to sequence DNA using nanopore sensors.
Nanopore sequencing is a unique, scalable technology that enables direct, real-time analysis of long DNA or RNA fragments. It works by monitoring changes to an electrical current as nucleic acids are passed through a protein nanopore. The resulting signal is decoded to provide the specific DNA or RNA sequence.
Oxford Nanopore was founded in Oxford Nanolabs by Dr.Gordon Sanghera, Dr.Spike Willcocks and Professor Hagan Bayley. Nanopore sequencing has been around since the 1990s, when Church et al. and Deamer and Akeson separately proposed that it is possible to sequence DNA using nanopore sensors.
Biotechnophysics: DNA Nanopore SequencingMelanie Swan
Biophysics (not merely bioengineering) is required to understand the fundamental mechanisms of biology in order to make technologies (bench and bioinformatic) for understanding them
A class of DNA sequencing techniques currently in active development is third-generation sequencing, commonly referred to as long-read sequencing. In comparison to second generation sequencing, also referred to as next generation sequencing, third generation sequencing technologies have the capacity to create noticeably longer reads.
Next generation-sequencing.ppt-convertedShweta Tiwari
The advance version, sequences the whole genome efficiently with high speed and high throughput sequencing at reduce cost is termed as Next Generation Sequencing (NGS) or massively parallel sequencing (MPS).
A new method of Nucleic Acid Sequencing using Nanotechnological Advances with Advantage of Single molecule sequencing, low cost and time requirement, easy to handle
Original Next Gen Seq Methods set of slides prepared for Technorazz Vibes 2016. There is also a shorter version.
This starts with an introduction to qPCR followed by an introduction to Library Complexity. Microarrays are discussed as well along with a very short introduction to FISH. Finally discussion of Next gen seq methods is done where generation of sequencers are discussed and a short discussion of the ILLUMINA protocol. Finally comparison of ILLUMINA amongst other 3rd gen sequencer, description of the standard pipeline and the omics technologies that have risen from this seq data.
Next Generation Sequencing (NGS) Is A Modern And Cost Effective Sequencing Technology Which Enables Scientists To Sequence Nucleic Acids At Much Faster Rate. In This Presentation, You Will Learn About What is NGS, Idea Behind NGS, Methodology And Protocol, Widely Adapted NGS Protocols, Applications And References For Further Study.
In a detail description of the two major blotting techniques. Right from its history to the result interpretation put forth in a concise way. Helps understand these protocols with ease.
Biotechnophysics: DNA Nanopore SequencingMelanie Swan
Biophysics (not merely bioengineering) is required to understand the fundamental mechanisms of biology in order to make technologies (bench and bioinformatic) for understanding them
A class of DNA sequencing techniques currently in active development is third-generation sequencing, commonly referred to as long-read sequencing. In comparison to second generation sequencing, also referred to as next generation sequencing, third generation sequencing technologies have the capacity to create noticeably longer reads.
Next generation-sequencing.ppt-convertedShweta Tiwari
The advance version, sequences the whole genome efficiently with high speed and high throughput sequencing at reduce cost is termed as Next Generation Sequencing (NGS) or massively parallel sequencing (MPS).
A new method of Nucleic Acid Sequencing using Nanotechnological Advances with Advantage of Single molecule sequencing, low cost and time requirement, easy to handle
Original Next Gen Seq Methods set of slides prepared for Technorazz Vibes 2016. There is also a shorter version.
This starts with an introduction to qPCR followed by an introduction to Library Complexity. Microarrays are discussed as well along with a very short introduction to FISH. Finally discussion of Next gen seq methods is done where generation of sequencers are discussed and a short discussion of the ILLUMINA protocol. Finally comparison of ILLUMINA amongst other 3rd gen sequencer, description of the standard pipeline and the omics technologies that have risen from this seq data.
Next Generation Sequencing (NGS) Is A Modern And Cost Effective Sequencing Technology Which Enables Scientists To Sequence Nucleic Acids At Much Faster Rate. In This Presentation, You Will Learn About What is NGS, Idea Behind NGS, Methodology And Protocol, Widely Adapted NGS Protocols, Applications And References For Further Study.
In a detail description of the two major blotting techniques. Right from its history to the result interpretation put forth in a concise way. Helps understand these protocols with ease.
whole genome analysis
history
needs
steps involved
human genome data
NGS
pyrosequencing
illumina
SOLiD
Ion torrent
PacBio
applications
problems
benefits
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles of a specific DNA sequence, known as probes.
This chapter provides an overview of DNA microarrays. Microarrays are a technology in which 1000’s of nucleic acids are bound to a surface and are used to measure the relative concentration of nucleic acid sequences in a mixture via hybridization and subsequent detection of the hybridization events. We first cover the history of microarrays and the antecedent technologies that led to their development. We then discuss the methods of manufacture of microarrays and the most common biological applications. The chapter ends with a brief discussion of the limitations of microarrays and discusses how microarrays are being rapidly replaced by DNA sequencing technologies.
The DNA microarray is a tool used to determine whether the DNA from a particular individual contains a mutation in genes like BRCA1 and BRCA2. The chip consists of a small glass plate encased in plastic. Some companies manufacture microarrays using methods similar to those used to make computer microchips.
Sequencing genes and genomes in biology. The most important technique available to the molecular biologist is DNA sequencing, by which the precise order of nucleotides in a piece of DNA can be determined
Microarray -types, DNA chip, Principle and application of microarray, Preparation of DNA Chip, Affymetrix chip, microarray in genomics and proteomics, advantages and limitations of microarray
Similar to Nanopore for dna sequencing by shreya (20)
1. A NANOPORE FOR DNA SEQUENCING
Guided and Checked By:
Dr. Prakash C. Jha Sir
Presented BY:
Shreya M .Modi
M.Phil. in Nano science
Central University of Gujarat.
shreyamodi20@gmail.com
1
http://www.upenn.edu/pennnews/sites/default/files/imagecache/large_news_photo/news/images/JR.jpg
3. INTRODUCTION
• Nanoporous materials consist of a regular
organic or inorganic framework supporting a
regular, porous structure.
• The size of the pores is generally 100
nanometers or smaller.
• Classified as Bulk
• Examples – Zeolites, A.Carbon, etc…
https://engineering.purdue.edu/ChE/Research/Areas/Nanotech/Images/Nanotech-02.jpg 3
7. DNA SEQUENCING
• DNA sequencing is the
process of determining
the precise order of
Nucleotides within a DNA
molecule.
• DNA sequencing may be
used to determine the
sequence of individual
genes, larger genetic
regions, full chromosomes
or entire genomes.
http://upload.wikimedia.org/wikipedia/commons/thumb/3/3d/Radioactive_Fluorescent_Seq.jpg/220px 7
Radioactive_Fluorescent_Seq.jpg
8. DNA SEQUENCING GENERATIONS
Then + Now Now Now + anticipated Anticipated
1st Gen 2nd Gen 2nd Gen Next
Sanger -parallised -single mol or electronic -single mol AND electronic
•Optical
•Optical
•Low •Single-molecule •Direct electrical (no optics)
•Amplification needed
throughput •Highly parallel •Single-molecule, highly parallel
•Highly parallel
•High cost •Cost similar •Transformation of workflow
•Improved cost and
•Accurate •New applications •Designed to broaden user base,
Throughput
•Broad user deliver step change in cost, power
•More centralised
base •Or electronic, •New applications
users
clonal
Helicos
GAII (Solexa/Illumina)
Pacific Biosciences
Sanger SOLiD (Agencourt/LIFE) Nanopores
Ion Torrent
FLX (454/Roche)
(LIFE Starlight)
Estimated cost of a human genome using these technologies
$70M $200k --- $50k ---- $20k --- 15k--- ?$5k - $?
8
12. WHAT IS A NANOPORE?
Nanopore = „very small hole‟
Electrical current flows through the hole
Introduce analyte of interest into the hole
identify “analyte” by the disruption or block to
Current electrical current
the
flow
12
13. APPLICATION OF NANOPORE
Adaptable protein nanopore:
Application
Specific
DNA Sequencing Proteins Polymers Small Molecules
Sensor array chip: many nanopores in parallel
Generic Platform
Electronic read-out system
13
14. NANO PORE FOR DNA SEQUENCING
• A nanopore is simply a small hole, of
the order of 1 nanometer in internal
diameter.
• Certain porous transmembrane
cellular proteins act as nanopores,
and nanopores have also been made
by etching a somewhat larger hole
• α-Hemolysin Nanopore
• MspA Nanopore
• Graphene Nanopore
14
https://pubs.acs.org/cen/_img/87/i10/8710sci2_DNA1.jpg
15. α-HEMOLYSIN
• TOXIN
• DNA or RNA strands can
translocate through the
pore of alpha-hemolysin,
producing the ionic current
blockades that reflect the
chemical structure of
individual strands
http://upload.wikimedia.org/wikipedia/en/thumb/8/82/The_process_of_hemolysis.png/450px-
15
The_process_of_hemolysis.png
16. ALPHA HEMOLYSIN
• A single nucleotide resolution has been
demonstrated for DNA hairpins raising the
prospect of creating a nanopore sensor
capable of reading the nucleotide
16
http://www.ks.uiuc.edu/~alek/HEMOLYSIN/stochasticSensor.jpg
17. DNA SEQUENCING BY MSPA
Mycobacterium smegmatis
17
http://ars.els-cdn.com/content/image/1-s2.0-S1368764612000180-gr1.jpg
18. Conti….
It resolve single-nucleotides in single-stranded DNA
when double-stranded DNA temporarily holds the
nucleotides in the pore constriction.
Passing DNA with a series of double-stranded
sections through MspA provides proof of principle of
a simple DNA sequencing method using a nanopore.
18
http://www.pnas.org/content/107/37/16060/F1.small.gif
19. • (A) The positive voltage attracts the negatively charged hairpin
DNA into the pore.
• (B) The DNA threads through the pore until the wider hairpin
duplex prevents further translocation.
• (C) After a few milliseconds the hairpin dissociates allowing for
complete translocation.
19
http://www.pnas.org/content/107/37/16060/F2.medium.gif
20. GRAPHENE FOR DNA SEQUENCING
Each nucleotide interacts with the nanopore to a varying
degree, resulting in a characteristic electronic signal for
each of the 4 nucleotides.
http://onlinelibrary.wiley.com/doi/10.1002/adfm.201002530/pdf 20
22. EDGE HYDROGENATION
When two H-bonds (dotted yellow lines) are formed simultaneously
between the nitrogen atom of a DNA nucleobase and two H atoms
attached to the graphene-edge.
For the sake of clarity, only relevant atoms from the edge hydrogenated
graphene electrodes and the DNA molecule have been visualized,
omitting water molecules, counter ions, and the silicon-nitride
membrane.
http://media.materialsviews.com/wp-content/uploads/2012/02/graphene-for-DNA-sequencing.gif 22
23. DETECTING A-T AND G-C BASE PAIRS
A-T base pairs to stretch more readily.
It can also discriminate between A-T and G-C base
pairs which is the first step towards sequencing DNA.
23
http://www.ks.uiuc.edu/Research/graphenepores/FIG/ATGC.png
24. Voltage-dependent Kinetics of DNA Transport
• The blocking is more effective at lower bias voltages.
• At low bias hydrophobic interaction is strong so DNA stick to
graphene membrane.
24
http://www.ks.uiuc.edu/Research/graphenepores/FIG/pot.png
25. ULTIMATELY: WILL WE SEQUENCE
EVERY SPECIES?
1995
2002 2005
2000
2002 2009
25
http://seedmagazine.com/interactive/genome/
26. CONCLUSION
• According to advancement and applications of
nanopores, it indicates that DNA sequencing
carried out by Nanopores are best Methods
than others.
26
27. REFERENCES
• http://www.ks.uiuc.edu/Research/graphenepores/
• https://pubs.acs.org/cen/science/87/8710sci2.html
• http://textbookofbacteriology.net/staph_3.html
• http://en.wikipedia.org/wiki/DNA_sequencing
• Faller M, et al. (2004) "The structure of a mycobacterial outer-
membrane channel." Science.
• Butler TZ, Pavlenok M, Derrington I, Niederweis M, Gundlach J
(2008). "Single-molecule DNA detection with an engineered MspA
protein nanopore." Proc. Natl. Acad. Sci. 106 (9): 20647-20652.
• Purnell R, Mehta K, Schmidt J (2008). Nucleotide identification and
orientation discrimination of DNA homopolymers immobilized in a
protein nanopore. Nano Letters 8 (9): 3029-3034
27
28. Conti…
• Church, G.M.; Deamer, D.W., Branton, D., Baldarelli, R., Kasianowicz,
J. (1998) "US patent # 5,795,782 (filed March 1995) Characterization
of individual polymer molecules based on monomer-interface
interaction".
• Kasianowicz, JJ; Brandin E, Branton D, Deamer DW (1996-11-26).
"Characterization of individual polynucleotide molecules using a
membrane channel.". Proc Natl Acad Sci USA 93 (24): 13770–3.
doi:10.1073/pnas.93.24.13770. PMC 19421. PMID 8943010.
• Manrao E, Derrington I, Pavlenok M, Niederweis M, Gundlach J
(2011). "Nucleotide discrimination with DNA immobilized in the
MspA nanopore." PLoS ONE 6
• Stoddart D, Heron A, Mikhailova E, Maglia G, Bayley H (2009).
"Single-nucleotide discrimination in immobilized DNA oglionucleoties
with a biological nanopore". Proc. Natl. Acad. Sci. USA 106: 7702– 28
7707.