Florida International University
College of Engineering
DNA SEQUENCING TECHNOLOGY
Name: Ahmad Nabil Abbas
Panther ID: 2651988
Dr. Nezih Pala
DNA:
Is a nucleic acid that contains the genetic
instructions used in the development and functioning
of all known living organisms and some viruses.
-DNA Store information and contains instructions to
construct cells
-DNA segments that store genetic information are
called GENES
-There are approximately 25,000 Genes in the Human
Genome
-In the Cell DNA is arranged into Chromosomes
DNA Composition:
-Two Polymers of units called NUCLEOTIDES
-Double Helix
-Backbone
Backbone made of Sugar and Phosphate
-Attached to the Sugar is one of four kinds of
Molecules:
adenine (A), cytosine (C), guanine (G) and thymine
(T)
-Base-pairing rule: A with T , C with G
http://www.youtube.com/watch?v=qy8dk5iS1f0
Bases Order in the Genome:
-Specific order for different chromosomes
-Humans are 99% alike
-DNA
DNA sequence Determines genetic characters of a
human
-These characters include any
y genetic
g
disease and/or
human property
-All creatures have the same structure but different
order
-Just like a strain of Bits
DNA SEQUENCING:
methods for determining the order of the nucleotide bases:
adenine guanine,
adenine,
guanine cytosine
cytosine, and thymine—in a molecule of DNA.
DNA
WHY?
-The DNA sequences making up any organism comprise the
basic blueprint for that organism (The Secret Of Life)
Molecular Medicine:
-Disease
Di
Gene
G
Identification
Id tifi ti
-Earlier detection of genetic predispositions to disease
-Drugs
Drugs designed to target specific gene products that cause
disease
-Pharmacogenomics "custom drugs”
-Replacement of defective genes for certain diseases
DNA Forensics
Human Gene Mutation
Agriculture and Breeding:
-Disease-,
Disease , insect
insect-,, and drought
drought-resistant
resistant crops
-Healthier, more productive, disease-resistant farm
animals
-More nutritious produce
-Edible vaccines incorporated into food products
DNA Sequencing Requirements:
-Fast
-Cheap
-Accurate
The Human Genome Project:
An International scientific research project with a primary goal
to determine the sequence of chemical base pairs which make
up DNA and to identify and map the approximately 20,000–
25,000 genes of the human genome from both a physical and
functional standpoint
-Started 1990 to 2003
-Parallel Sequencing
-sequencing was performed in universities and research
centers from the United States, the United Kingdom, Japan,
France, Germany, China, Canada, and New Zealand
http://www.youtube.com/watch?v=-gVh3z6MwdU
The X Prize:
The $10 million X PRIZE for Genomics prize purse
will be awarded to the first Team that can build a
device and use it to sequence 100 human genomes
within 10 days or less, with an accuracy of no more
than one error in every 100,000 bases sequenced,
with sequences accurately covering at least 98% of
the genome,
genome and at a recurring cost of no more than
$10,000 per genome.
DNA Sequencing Methods:
-1953, James Watson and Francis Crick concluded
that DNA contained the "stuff of life“…
1) Maxam-Gilbert Method (1976):
-Based on unique
q
chemical reactions on one or two
of the bases.
- Steps:
*Chemical treatments to break the fragments
*Gel
Gel Electrophoresis
*Autoradiography
- Disadvantages: Technical Complexity
Complexity, Use of
Hazardous Materials
2) Chain-Termination Method (Sanger):
-More efficient, Less toxic chemicals than M-G method.
Steps:
-Four
F
sett off R
Reactions
ti
are needed
d d
-In each of them a copy of the DNA needed to be sequenced, an
extra supply of A, G, C and T, dideoxynucleotides (ddATP,
ddGTP, ddCTP, or ddTTP) which are the chain-terminating
nucleotides, a Primer and a DNA Polymerase.
-Since
Since each copy will be cut in a different position. By time all
the possible cuts will happen and various lengths of fragments
will be present
-The
The DNA is then denatured and the contents of each reaction
will be placed on a separate lane in the Polyacrylmide gel to
separate defferent lengths and UV light will show the tagged
base accordingly.
http://www.youtube.com/watch?v=aPN8LP4YxPo&feature=related
3) Dye-Terminator Method
-Dye-terminator sequencing utilizes labelling of the
chain terminator ddNTPs, which permits sequencing
in a single reaction, rather than four reactions as in
the labelled
labelled-primer
primer method.
-each of the four dideoxynucleotide chain
terminators is labelled with fluorescent dyes,
y , each of
which with different wavelengths of fluorescence
and emission
htt //
http://www.youtube.com/watch?v=dUjMf2ZezIw
t b
/
t h? dUjMf2Z I
Advanced Methods:
-The X Prize has led to a huge number of
contributions
-Two methods according to researchers are
approaching
hi
promising
i i
results
lt iin tterms off
practicality.
- Nanopores And Real-time
Real time detection of dNTP
incorporation
Nanopores DNA sequencing:
-A nanopore is a small opening in the order of couple of
nanometers diameter separating two chambers with an ability
to have single molecules pass through it at a given time
-The main idea is to have two ionic solutions in two chambers
separated
t d by
b a nanopore
-The underlying principle of nanopore sequencing is that a
single-stranded DNA or RNA molecule can be
electrophoretically driven through a nano-scale pore in such a
way that the molecule traverses the pore in strict linear
sequence
-Solid state nanopores with probes that serve as atomic scale
electrodes whose termini are on opposite sides of the
nanopore.
p
A voltage
g bias between these probes
p
will induce an
electronic current stimulated by quantum mechanical tunneling
to flow from one side of the nanopore to the other. The current,
which will be sensed by an external circuit, will be modulated
by the individual nucleotide bases as ssDNA molecules
translocate through the nanopore.
Nanopores DNA sequencing:
Controlling the flow of the ssDNA (DNA
translocation):
-A certain voltage is applied across the chambers
will
ill lead
l d th
the DNA to
t fl
flow ffrom th
the negative
ti
polarity
l it
to the positive one with a certain speed according to
the voltage
g (120
(
mV is currently
y being
g used).
)
-Also application of a certain viscous material at the
pore itself can contribute to the speed control of
DNA translocation.
Current Achievements:
-Length and shape Detection
-DNA/SWNT Hybrid
E
Expected
t dA
Achievements:
hi
t
1 MBs
Current Challenges:
-Improve nanopore surfaces to reduce non-specific adsorption,
pore clogging, and electrical noise
-Fabricate and test a nanopore detector articulated with
integrated probes for molecular identification
-Develop
p new enzymatic
y
methods to better control and limit the
rate of DNA translocation through articulated nanopores
-Develop algorithms for signal feature detection and base
identification from articulated nanopores
Nanopore Technology (Alternative Method):
-Developed by NABsys
-Parallel pores
-Hybrid
Hybrid sequencing
-Requires amplification (Major Disadvantage)
-Applicable
Applicable with the current technology
htt //
http://www.youtube.com/watch?v=HV0aWVrDM2U
t b
/
t h? HV0 WV DM2U
Real-Time Detection of dNTP incorporation:
-Developed by VisiGene, TX
-Showed the most promising results (patents in europe and the
US)
-Currently
Currently achieved 1 MBs
Method:
-Uses Fluorescently tagged PP1
-The time-dependent fluorescent signals emitted from each
complex
l
are monitored
it
d and
d analyzed
l
d tto d
determine
t
i
DNA
sequence information.
-Energy
gy transfers from donor to acceptor,
p , stimulating
g emission
-Create massively parallel arrays of nanomachines engineered
to act as molecular sensors of base identity during dNTP
incorporation
-Direct analysis - no cloning or amplification
Detection System:
Video Visualization of the technology:
http://www.youtube.com/watch?v=XLjzBTpk5J4&feature=related
References:
-http://www.nd.edu/~aseriann/maxam.html
-http://en.wikipedia.org/wiki/Human_Genome_Project
-http://en.wikipedia.org/wiki/DNA
-http://genomics.xprize.org/archon-x-prize-for-genomics
-http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Obenrader/sanger_method_page.htm
-http://en.wikipedia.org/wiki/Sequencing_by_hybridization
http://en wikipedia org/wiki/Sequencing by hybridization
-http://en.wikipedia.org/wiki/DNA_sequencing#cite_ref-22
-http://golgi.harvard.edu/branton/projects-NanoporeSequencing.htm
-VisiGene Biotech Inc, Real-Time DNA sequencing
-Mary Hughes, Optical Absorption of DNA/Carbon Nanotube Structure, 2007
-Daniel Fologea, DNA Conformation and Base Number Simultaneously determined in a nanopore, 2007.
-Fernando Albertorio, Base dependent DNA–carbon nanotube interactions: activation enthalpies and
assembly–disassembly control, 2009