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FOUNDED BY BRITTANY L ROARK

FOUNDED BY BRITTANY L ROARK. DNA Sequencing History. The structure of DNA was established as a double helix in 1953. Frederick Sanger developed rapid DNA sequencing methods at the MRC Centre in Cambridge, UK and published a method for "DNA sequencing with chain-terminating inhibitors" in 1977.

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FOUNDED BY BRITTANY L ROARK

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  1. FOUNDED BY BRITTANY L ROARK

  2. DNA Sequencing History • The structure of DNA was established as a double helix in 1953. Frederick Sanger developed rapid DNA sequencing methods at the MRC Centre in Cambridge, UK and published a method for "DNA sequencing with chain-terminating inhibitors" in 1977. • Walter Gilbert and Allan Maxam at Harvard also developed sequencing methods. In 1973, Gilbert and Maxam reported the sequence of 24 base pairs using a method known as wandering-spot analysis. • The first full DNA genome to be sequenced was in 1977. Medical Research Council scientists found that the complete DNA sequence of the Epstein-Barr virus is170 thousand base-pairs long in 1984. • In 1986, Leroy E. Hood's laboratory at the California Institute of Technology announced the first semi-automated DNA sequencing machine. Then Applied Biosystems' marketed the first fully automated sequencing machine, the ABI 370, in 1987 and then Dupont's Genesis 2000 which used a novel fluorescent labeling technique which enabled all four dideoxynucleotides to be identified in a single lane. • In 1995, Craig Venter, Hamilton Smith, and colleagues at The Institute for Genomic Research (TIGR) published the first complete genome of a free-living organism, the bacterium Haemophilus influenzae. The circular chromosome contains 1,830,137 bases and its publication in the journal Science marked the first published use of whole-genome shotgun sequencing. • By 2001, shotgun sequencing methods had been used to produce a draft sequence of the human genome. • In 1996, Pål Nyrén and his student Mostafa Ronaghi at the Royal Institute of Technology in Stockholm published their method of pyrosequencing. • Pascal Mayer and Laurent Farinelli submitted patents to the World Intellectual Property Organization describing DNA colony sequencing in 1996. • Lynx Therapeutics published and marketed "Massively parallel signature sequencing", or MPSS, in 2000.

  3. Dideoxy Chain Termination Method • DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. • Dideoxy Chain Termination Method was created by Fredrick Sanger Gel Electrophoresis

  4. Dideoxy (Sanger) Method • There are four steps: • Denaturation • Primer attachment and extension of bases • Termination • Gel electrophoresis • Run four separate reactions each with different ddNTPs (dideoxynucleotides) • Run on a gel in four separate lanes • Read the gel from the bottom up

  5. Dideoxy (Sanger) Method Cont. • The chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleosidetriphosphates (dNTPs), and modified di-deoxynucleosidetriphosphates (ddNTPs) (which terminate DNA strand elongation). ddNTPs lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, which causes DNA polymerase to stop extension of DNA when a modified ddNTP is incorporated. "The ddNTPs may be radioactively or fluorescently labeled for detection in automated sequencing machines." • The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. Only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) is added to each reaction. The other three nucleotides are ordinary ones. "Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis." • In the 1977 original publication, the formation of base-paired loops of ssDNA was a cause of serious difficulty in resolving bands at some locations. The DNA bands may then be visualized by autoradiography or UV light and the DNA sequence can be directly read off the X-ray film or gel image.

  6. The Human Genome Project • The Human Genome Project (HGP) was a research program that had a goal of the complete mapping and understanding of all the genes of humans. All our genes together are known as our "genome." • The HGP has revealed that there are approximately 20,500 human genes. The completed human sequence can now identify their locations. This product of the HGP has given us detailed information about the structure, organization and function of the complete set of human genes. • In February of 2001, The International Human Genome Sequencing Consortium published the first draft of the human genome in the journal Nature with the sequence of the entire genome's 3 billion base pairs about 90% complete. The full sequence was completed and published in April of 2003

  7. Current Uses (Sanger Method) • single nucleotide polymorphism (SNP) detection • single-strand conformation polymorphism (SSCP) • hetroduplex analysis • short tandem repeat (STR) analysis • most critical step in studying features of the genome: resolving DNA fragments according to differences in size and/or conformation

  8. Current Uses (HGP) • If other disease-related genes are isolated, scientists can begin to understand the structure and pathology of other disorders like heart disease, cancer, and diabetes. Which would lead to the creation of better medicines and cures. • "Current and potential applications of genome research will address national needs in molecular medicine, waste control and environmental cleanup, biotechnology, energy sources, and risk assessment." - Human Genome Project Archive

  9. Bioethical Considerations • "The Ethical, Legal, and Social Implications (ELSI) program focused on the possible consequences of genomic research in four main areas: • Privacy and fairness in the use of genetic information, including the potential for genetic discrimination in employment and insurance. • The integration of new genetic technologies, such as genetic testing, into the practice of clinical medicine. • Ethical issues surrounding the design and conduct of genetic research with people, including the process of informed consent. • The education of healthcare professionals, policy makers, students, and the public about genetics and the complex issues that result from genomic research."

  10. Single-nucleotide polymorphism(SNP) • SNPs have been used in genome-wide association studies (GWAS). For example, high-resolution markers in gene mapping related to diseases or normal traits. The knowledge of SNPs will help in understanding how drugs act in individuals with different genetic variants. • A wide range of human diseases like Sickle–cell anemia, β Thalassemia and Cystic fibrosis result from SNPs. Diseases with different SNPs may become new targets for drug therapy. Some SNPs are associated with the metabolism of different drugs.

  11. Sources • http://www.nature.com/scitable/topicpage/dna-sequencing-technologies-690 • http://en.wikipedia.org/wiki/DNA_sequencing • http://chemistry.montana.edu/~martint/BCHM444/DNA%20Sequencing%20Methods.ppt • http://www.genome.gov/12011238 • http://en.wikipedia.org/wiki/Sanger_sequencing • http://ghr.nlm.nih.gov/handbook/hgp/elsi • http://en.wikipedia.org/wiki/Single_nucleotide_polymorphism#Use_and_importance

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