DNA sequencing: Importance
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DNA sequencing: Importance

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DNA sequencing: Importance

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1. DNA sequencing: Importance The DNA sequences making up any organism comprise the basic blueprint for that organism

3. 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.

4. 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.

5. 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.

6. 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??

7. Maxam-Gilbert ?chemical? method

8. 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)

9. 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

10. ddNTPs are analagous to faulty LEGOs, DNA Analysis: DNA Sequencing

19. 10_07_1_enzym.dideoxy.jpg 10_07_1_enzym.dideoxy.jpg10_07_1_enzym.dideoxy.jpg

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25. http://www.dnai.org/b/index.html DNA Analysis: DNA Sequencing

31. 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 ?m 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.

32. Pyrosequencing


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