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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

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DNA sequencing: Importance

  • The DNA sequences making up any organism comprise the basic blueprint for that organism


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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…


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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.


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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.


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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.


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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??

DNA sequencing methodologies: ca. 1977



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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)

versus “synthesis-based” methods


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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


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DNA Analysis: DNA Sequencing

  • ddNTPs are analagous to faulty LEGOs,

Faulty LEGOs lack the

little pegs and nothing can

stack on them – thus,

they ‘terminate’ the stack

Normal LEGOs have

little pegs that allow

them to stack




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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


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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.


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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.


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Pyrosequencing

Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001


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Pyrosequencing - Solid Phase

Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001


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Pyrogram

Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001



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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


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Alternatives to Dye Terminator Sequencing

  • Others:

    • Microarray sequencing – aka sequencing by hybridization


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Alternatives to Dye Terminator Sequencing

  • Others:

    • Nanopore sequencing