Đề tài DNA sequencing?

Group: Nguyen Trung Tinh Nguyen Ngoc Tai Le Tran Danh Nghia 	Le Minh Diep	 Pham Tai Nhi Pham Hoang NamWhat is DNA sequencing?DNA sequencing is the determination ofthe precise sequence of nucleotides in asample of DNA.DNA sequencing: Importance	The DNA sequences making up anyorganism comprise the basic blueprint for that organism. Potential benefitsMolecular 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 genotypeThe Human Genome Project (and others) Potential benefitsBioarchaeology, 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 benefitsDNA 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 benefitsAgriculture, 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 productsNew environmental cleanup uses for plants like tobacco.The Human Genome Project (and others) DNA sequencing methodologies: ca. 1977Maxam-Gilbert base modification by general and specific chemicals.depurination or depyrimidination.single-strand excision.not amenable to automationSangerDNA replication.substitution of substrate with chain-terminator chemical.more efficientautomation??Maxam-Gilbert ‘chemical’ methodversus “synthesis-based” methodsFred Sanger: Nobel Prize 1980Instead of taking a complete sequence and breaking it down, build DNA sequences up and analyze steps along the wayThey key to this process: dideoxynucleotides (ddNTPs)What to label for visualization?Primers?Disadvantages of primer-labels:four reactionstediouslimited to certain regions, custom oligos orlimited to cloned inserts behind ‘universal’ priming sites.Advantages: it works Solution:labeled “terminators” - ddNTPsddNTPs are analagous to faulty LEGOsDNA Analysis: DNA SequencingNormal LEGOs have little pegs that allow them to stackFaulty LEGOs lack the little pegs and nothing can stack on them – thus, they ‘terminate’ the stackThis is great butWouldn’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 377Trace 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 Sequencing454 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 bp454 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. PyrosequencingRonaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001Pyrosequencing - Solid PhaseRonaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001PyrogramRonaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001454 LifeSciences Sequencer454 LifeSciences SequencerAdvantagesFast, accurateGreat for small, simple genomesDisadvantagesReads only ~100 – 200 bpCrappy for large complex genomes (like ours)Homopolymer stretches (8+) are difficult to read Alternatives to Dye Terminator SequencingOthers:Microarray sequencing – aka sequencing by hybridizationAlternatives to Dye Terminator SequencingOthers:Nanopore sequencinggood bye teacher and classreference materials:www.ebook.edu.vnwww.ebook4u.vnwww.sinhhocvietnam.comen.wikipedia.org