【文章內(nèi)容簡介】 itycontrol measures. . Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2023 ELSI Issues (cont.) ? Uncertainties associated with gene tests for susceptibilities and plex conditions (., heart disease, diabetes, and Alzheimer’s disease). ? Fairness in access to advanced genomic technologies. ? Conceptual and philosophical implications regarding human responsibility, free will vs geic determinism, and concepts of health and disease. ? Health and environmental issues concerning geically modified (GM) foods and microbes. ? Commercialization of products including property rights (patents, copyrights, and trade secrets) and accessibility of data and materials. . Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2023 Beyond the HGP: What’s Next? HapMap Systems Biology Exploring Microbial Genomes for Energy and the Environment Chart geic variation within the human genome Genomes to Life: A DOE Systems Biology Program Exploring Microbial Genomes for Energy and the Environment Goals ? identify the protein machines that carry out critical life functions ? characterize the gene regulatory works that control these machines ? characterize the functional repertoire of plex microbial munities in their natural environments ? develop the putational capabilities to integrate and understand these data and begin to model plex biological systems HapMap An NIH program to chart geic variation within the human genome ? Begun in 2023, the project is a 3year effort to construct a map of the patterns of SNPs (single nucleotide polymorphisms) that occur across populations in Africa, Asia, and the United States. ? Consortium of researchers from six countries ? Researchers hope that dramatically decreasing the number of individual SNPs to be scanned will provide a shortcut for identifying the DNA regions associated with mon plex diseases ? Map may also be useful in understanding how geic variation contributes to responses in environmental factors Dideoxy Sequencing ? Dideoxy sequencing (also called chain termination or Sanger method) uses an enzymatic procedure to synthesize DNA chains of varying lengths, stopping DNA replication at one of the four bases and then determining the resulting fragment lengths. Each sequencing reaction tube (T, C, G, and A) in the diagram contains – a DNA template, a primer sequence, and a DNA polymerase to initiate synthesis of a new strand of DNA at the point where the primer is hybridized to the template。 – the four deoxynucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) to extend the DNA strand。 – one labeled deoxynucleotide triphosphate (using a radioactive element or dye)。 and – one dideoxynucleotide triphosphate, which terminates the growing chain wherever it is incorporated. Tube A has didATP, tube C has didCTP, etc. Dideoxy Sequencing ? For example, in the A reaction tube the ratio of the dATP to didATP is adjusted so that each tube will have a collection of DNA fragments with a didATP incorporated for each adenine position on the template DNA fragments. The fragments of varying length are then separated by electrophoresis and the positions of the nucleotides analyzed to determine sequence. The fragments are separated on the basis of size, with the shorter fragments moving faster and appearing at the bottom of the gel. Sequence is read from bottom to top Dideoxy Sequencing Polyacrylamide gel with small pores is used to fractionate singlestranded DNA. In the size range 10 to 500 nucleotides, DNA molecules that differ in size by only a single nucleotide can be separated Lane 1 partial replicas terminating in G Lane 2 partial replicas terminating in A Lane 3 partial replicas terminating in T Lane 4 partial replicas terminating in C Alberts et al. Molecular Biology of the Cell. Brown. Genomes 2 Capillary Electrophoresis ? Technique bines the use of gelfilled capillary tubes with a unique laser scanning system to sequence each of the four different types of bases adenine, cytosine, guanine, and thymine in a sample of DNA. ? Capillary array electrophoresis cuts the time of the Sanger dideoxy method down by replacing the slab with hundreds of tiny gelfilled capillaries, about 100 microns (four thousandths of an inch) in internal diameter, that can be bundled into a single array for automated detection. Capillary Electrophoresis ? The dideoxy method was improved by the use of fluorescent labels. The primer is synthesized and split into four batches, each of which is labeled with a different fluorescent dye. ? Each dye labeled primer is used in a sequencing reaction with one of the dideoxynucleotides. ? The reaction products are pooled and analysed in a single lane of a sequencing gel. A fourcolour fluorescence detector monitors the DNA as it migrates to the bottom of the gel. The fluorescence signature is used to identify the terminal nucleotide. Capillary Electrophoresis ? A method was then developed based on this where fluorescent dideoxynucleotides were used in a single sequencing reaction. The fragments are then separated in a single lane of a sequencing gel and identified by the fluorescent signature. ? The limitations of gel electrophoresis soon became apparent. Gels take a long time to run and have a limited reproducibility. An automated method was much more desirable, but the automation of gels requires plex robotic handling. ? The use of capillaries allows much higher electrical fields to be used making the separations faster. Flexible capillaries are also easily incorporated into an automated instrument making sequencing cheap, fast and efficient. ? However a single capill