Sinh hoc phan tu

Group:
Nguyen Trung Tinh
Nguyen Ngoc Tai
Le Tran Danh Nghia
Le Minh Diep
Pham Tai Nhi
Pham Hoang Nam
What is DNA sequencing?
DNA sequencing is the determination of
the precise sequence of nucleotides in a
sample of DNA.
DNA sequencing: Importance
The DNA sequences making up any
organism comprise the basic blueprint for
that organism.



Potential benefits
Molecular medicine
 Improved diagnosis of disease.
Disease gene identification will lead to more accurate diagnosis.
 Earlier detection of genetic predispositions to disease.
Will be able to assess risk for certain diseases, e.g. cancer, Type II diabetes, heart disease.
 Rational drug design.
Drugs designed to target specific gene products that cause disease.
 Gene therapy and control systems for drugs.
Replacement of defective genes for certain diseases.
 Pharmacogenomics "custom drugs”.
Drug therapy based on ones genotype…

The Human Genome Project (and others)
Potential benefits
Bioarchaeology, anthropology, evolution, and human migration.
Study evolution through germline mutations in lineages.
Study migration of different population groups based on female genetic inheritance.
Study mutations on the Y chromosome to trace lineage and migration of males.
Compare breakpoints in the evolution of mutations with ages of populations and historical events.

The Human Genome Project (and others)
Potential benefits
DNA forensics (identification)
Identify potential suspects whose DNA may match evidence left at crime scenes.
Exonerate persons wrongly accused of crimes.
Identify crime and catastrophe victims.
Establish paternity and other family relationships.
Identify endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers).
Detect bacteria and other organisms that may pollute air, water, soil, and food.
Determine pedigree for seed or livestock breeds.

The Human Genome Project (and others)
Potential benefits
Agriculture, livestock breeding, and bioprocessing
Disease-, insect-, and drought-resistant crops.
Healthier, more productive, disease-resistant farm animals.
More nutritious produce .
Biopesticides.
Edible vaccines incorporated into food products
New environmental cleanup uses for plants like tobacco.

The Human Genome Project (and others)
DNA sequencing methodologies:
ca. 1977
Maxam-Gilbert
base modification by general and specific chemicals.
depurination or depyrimidination.
single-strand excision.
not amenable to automation
Sanger
DNA replication.
substitution of substrate with chain-terminator chemical.
more efficient
automation??
Maxam-Gilbert ‘chemical’ method
versus “synthesis-based” methods
Fred Sanger: Nobel Prize 1980
Instead of taking a complete sequence and breaking it down, build DNA sequences up and analyze steps along the way
They key to this process: dideoxynucleotides (ddNTPs)
What to label for visualization?
Primers?
Disadvantages of primer-labels:
four reactions
tedious
limited to certain regions, custom oligos or
limited to cloned inserts behind ‘universal’ priming sites.
Advantages: it works
Solution:
labeled “terminators” - ddNTPs
ddNTPs are analagous to faulty LEGOs
DNA Analysis: DNA Sequencing
Normal LEGOs have
little pegs that allow
them to stack
Faulty LEGOs lack the
little pegs and nothing can
stack on them – thus,
they ‘terminate’ the stack
This is great but…

Wouldn’t it be great to run everything in one lane?
Save space and time, more efficient

Fluorescently label the ddNTPs so that they each appear a different color
“virtual autorad” - real-time DNA sequence output from ABI 377
Trace files (dye signals) are analyzed and bases called to create chromatograms.

Chromatograms from opposite strands are reconciled with software to create double-stranded sequence data.
Alternatives to Dye Terminator Sequencing
454 Sequencing is a massively-parallel sequencing-by-synthesis (SBS) system
capable of sequencing roughly 20 megabases (20,000,000 bp) of raw DNA sequence per 4.5-hour run
Compare to best dye terminator sequencing rig today :ABI 3730xl
(192 capillaries x ~1000 bp) in 5 hrs (2 2.5 hr runs) = 196,000 bp
454 sequencing relies on fixing nebulized and adapter-ligated DNA fragments to small DNA-capture beads in a water-in-oil emulsion.
DNA is fixed to these beads is then amplified by PCR.
Each DNA-bound bead is placed into a ~44 μm well on a PicoTiterPlate, a fiber optic chip. A mix of enzymes such as polymerase, ATP sulfurylase, and luciferase are also packed into the well.
The four nucleotides (TAGC) are washed in series over the PicoTiterPlate.
If a nucleotide complementary to the template strand flows into a well, the polymerase extends the existing DNA strand by adding nucleotide(s).
Addition in a reaction that generates a light signal that is recorded by acamera in the instrument.
Pyrosequencing
Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001
Pyrosequencing - Solid Phase
Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001
Pyrogram
Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001
454 LifeSciences Sequencer
454 LifeSciences Sequencer
Advantages
Fast, accurate
Great for small, simple genomes
Disadvantages
Reads only ~100 – 200 bp
Crappy for large complex genomes (like ours)
Homopolymer stretches (8+) are difficult to read
Alternatives to Dye Terminator Sequencing
Others:
Microarray sequencing – aka sequencing by hybridization

Alternatives to Dye Terminator Sequencing
Others:
Nanopore sequencing

THE END
good bye teacher and class
reference materials:
www.ebook.edu.vn
www.ebook4u.vn
www.sinhhocvietnam.com
en.wikipedia.org