Download DNA Sequencing: Importance

DNA sequencing: Importance
• The DNA sequences making up any organism comprise
the basic blueprint for that organism
The Human Genome Project (and others)
• 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
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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.
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 chainterminator 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
DNA Analysis: DNA Sequencing
• ddNTPs are analagous to faulty LEGOs,
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
10_07_1_enzym.dideoxy.jpg
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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
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DNA Analysis: DNA Sequencing
• http://www.dnai.org/b/index.html
“virtual autorad” - real-time DNA sequence output from ABI 377
1. Trace files (dye signals) are analyzed and
bases called to create chromatograms.
2. Chromatograms from opposite strands are
reconciled with software to create doublestranded sequence data.
Alternatives to Dye Terminator Sequencing
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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 4.5hour 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 DNAcapture 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