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1、The Human Genome Project/sci/techresources/Human_Genome/education/images.shtml廣州網(wǎng)站建設(shè) IntroductionUntil the early 1970s, DNA was the most difficult cellular molecule for biochemists to analyze.DNA is now the easiest molecule to analyze we can now isolate a specific region of the genome, produce a vir

2、tually unlimited number of copies of it, and determine its nucleotide sequence overnight.Molecular Biology Of The Cell. Alberts et al. 491-495IntroductionAt the height of the Human Genome Project, sequencing factories were generating DNA sequences at a rate of 1000 nucleotides per second 24/7.Techni

3、cal breakthroughs that allowed the Human Genome Project to be completed have had an enormous impact on all of biology.Molecular Biology Of The Cell. Alberts et al. 491-495Human Genome ProjectGoals: identify all the approximate 30,000 genes in human DNA, determine the sequences of the 3 billion chemi

4、cal base pairs that make up human DNA, store this information in databases, improve tools for data analysis, transfer related technologies to the private sector, and address the ethical, legal, and social issues (ELSI) that may arise from the project. Milestones: 1990: Project initiated as joint eff

5、ort of U.S. Department of Energy and the National Institutes of Health June 2000: Completion of a working draft of the entire human genome (covers 90% of the genome to a depth of 3-4x redundant sequence) February 2001: Analyses of the working draft are published April 2003: HGP sequencing is complet

6、ed and Project is declared finished two years ahead of scheduleU.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003http:/www.sanger.ac.uk/HGP/overview.shtmlWhat does the draft human genome sequence tell us? By the Numbers The human genome contains 3 billion

7、 chemical nucleotide bases (A, C, T, and G). The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene being dystrophin at 2.4 million bases. The total number of genes is estimated at around 30,000-much lower than previous estimates of 80,000 to 140,000. Almo

8、st all (99.9%) nucleotide bases are exactly the same in all people. The functions are unknown for over 50% of discovered genes.U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003What does the draft human genome sequence tell us?How Its Arranged The human g

9、enomes gene-dense urban centers are predominantly composed of the DNA building blocks G and C. In contrast, the gene-poor deserts are rich in the DNA building blocks A and T. GC- and AT-rich regions usually can be seen through a microscope as light and dark bands on chromosomes. Genes appear to be c

10、oncentrated in random areas along the genome, with vast expanses of noncoding DNA between. Stretches of up to 30,000 C and G bases repeating over and over often occur adjacent to gene-rich areas, forming a barrier between the genes and the junk DNA. These CpG islands are believed to help regulate ge

11、ne activity. Chromosome 1 has the most genes (2968), and the Y chromosome has the fewest (231). U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003What does the draft human genome sequence tell us? The Wheat from the Chaff Less than 2% of the genome codes

12、for proteins. Repeated sequences that do not code for proteins (junk DNA) make up at least 50% of the human genome. Repetitive sequences are thought to have no direct functions, but they shed light on chromosome structure and dynamics. Over time, these repeats reshape the genome by rearranging it, c

13、reating entirely new genes, and modifying and reshuffling existing genes. The human genome has a much greater portion (50%) of repeat sequences than the mustard weed (11%), the worm (7%), and the fly (3%).U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003

14、What does the draft human genome sequence tell us? How the Human Compares with Other Organisms Unlike the humans seemingly random distribution of gene-rich areas, many other organisms genomes are more uniform, with genes evenly spaced throughout. Humans have on average three times as many kinds of p

15、roteins as the fly or worm because of mRNA transcript alternative splicing and chemical modifications to the proteins. This process can yield different protein products from the same gene. Humans share most of the same protein families with worms, flies, and plants; but the number of gene family mem

16、bers has expanded in humans, especially in proteins involved in development and immunity. Although humans appear to have stopped accumulating repeated DNA over 50 million years ago, there seems to be no such decline in rodents. This may account for some of the fundamental differences between hominid

17、s and rodents, although gene estimates are similar in these species. Scientists have proposed many theories to explain evolutionary contrasts between humans and other organisms, including those of life span, litter sizes, inbreeding, and genetic drift. U.S. Department of Energy Genome Programs, Geno

18、mics and Its Impact on Science and Society, 2003What does the draft human genome sequence tell us? Variations and Mutations Scientists have identified about 3 million locations where single-base DNA differences (SNPs) occur in humans. This information promises to revolutionize the processes of findi

19、ng chromosomal locations for disease-associated sequences and tracing human history. The ratio of germline (sperm or egg cell) mutations is 2:1 in males vs females. Researchers point to several reasons for the higher mutation rate in the male germline, including the greater number of cell divisions

20、required for sperm formation than for eggs. U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003What does the draft human genome sequence tell us?Led to the discovery of whole new classes of proteins and genes, while revealing that many proteins have been m

21、uch more highly conserved in evolution than had been suspected.Provided new tools for determining the functions of proteins and of individual domains within proteins, revealing a host of unexpected relationships between them. Molecular Biology Of The Cell. Alberts et al. 491-495What does the draft h

22、uman genome sequence tell us?By making large amounts of protein available, it has yielded an efficient way to mass produce protein hormones and vaccinesDissection of regulatory genes has provided an important tool for unraveling the complex regulatory networks by which eukaryotic gene expression is

23、controlled.Molecular Biology Of The Cell. Alberts et al. 491-495How does the human genome stack up? Gene number, exact locations, and functions Gene regulation DNA sequence organization Chromosomal structure and organization Noncoding DNA types, amount, distribution, information content, and functio

24、ns Coordination of gene expression, protein synthesis, and post-translational events Interaction of proteins in complex molecular machines Predicted vs experimentally determined gene function Evolutionary conservation among organisms Protein conservation (structure and function) Proteomes (total pro

25、tein content and function) in organisms Correlation of SNPs (single-base DNA variations among individuals) with health and disease Disease-susceptibility prediction based on gene sequence variation Genes involved in complex traits and multigene diseases Complex systems biology including microbial co

26、nsortia useful for environmental restoration Developmental genetics, genomics Future Challenges: What We Still Dont KnowU.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003Anticipated Benefits of Genome ResearchMolecular Medicine improve diagnosis of diseas

27、e detect genetic predispositions to disease create drugs based on molecular information use gene therapy and control systems as drugs design “custom drugs” (pharmacogenomics) based on individual genetic profiles Microbial Genomics rapidly detect and treat pathogens (disease-causing microbes) in clin

28、ical practice develop new energy sources (biofuels) monitor environments to detect pollutants protect citizenry from biological and chemical warfare clean up toxic waste safely and efficientlyU.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003Risk Assessme

29、nt evaluate the health risks faced by individuals who may be exposed to radiation (including low levels in industrial areas) and to cancer-causing chemicals and toxinsBioarchaeology, Anthropology, Evolution, and Human Migration study evolution through germline mutations in lineages study migration o

30、f different population groups based on maternal 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 U.S. Department of Energy Genome Programs, Genomics and Its Impact

31、on Science and Society, 2003Anticipated Benefits of Genome Research-cont.DNA Identification (Forensics) 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 famil

32、y 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 match organ donors with recipients in transplant programs determine pedigree for seed or

33、livestock breeds authenticate consumables such as caviar and wineU.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003Anticipated Benefits of Genome Research-cont.Agriculture, Livestock Breeding, and Bioprocessing grow disease-, insect-, and drought-resistan

34、t crops breed healthier, more productive, disease-resistant farm animals grow more nutritious produce develop biopesticides incorporate edible vaccines incorporated into food products develop new environmental cleanup uses for plants like tobacco U.S. Department of Energy Genome Programs, Genomics a

35、nd Its Impact on Science and Society, 2003Anticipated Benefits of Genome Research-cont.Anticipated Benefits: improved diagnosis of disease earlier detection of genetic predispositions to disease rational drug design gene therapy and control systems for drugs personalized, custom drugs Medicine and t

36、he New GeneticsU.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003Gene Testing Pharmacogenomics Gene TherapyELSI: Ethical, Legal, and Social Issues Privacy and confidentiality of genetic information. Fairness in the use of genetic information by insurers,

37、employers, courts, schools, adoption agencies, and the military, among others. Psychological impact, stigmatization, and discrimination due to an individuals genetic differences. Reproductive issues including adequate and informed consent and use of genetic information in reproductive decision makin

38、g. Clinical issues including the education of doctors and other health-service providers, people identified with genetic conditions, and the general public about capabilities, limitations, and social risks; and implementation of standards and qualitycontrol measures.U.S. Department of Energy Genome

39、Programs, Genomics and Its Impact on Science and Society, 2003ELSI Issues (cont.) Uncertainties associated with gene tests for susceptibilities and complex conditions (e.g., heart disease, diabetes, and Alzheimers disease). Fairness in access to advanced genomic technologies. Conceptual and philosop

40、hical implications regarding human responsibility, free will vs genetic determinism, and concepts of health and disease. Health and environmental issues concerning genetically modified (GM) foods and microbes. Commercialization of products including property rights (patents, copyrights, and trade se

41、crets) and accessibility of data and materials. U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003Beyond the HGP: Whats Next?HapMapSystems BiologyExploring Microbial Genomes for Energy and the EnvironmentChart genetic variation within the human genomeGeno

42、mes to Life: A DOE Systems Biology ProgramExploring Microbial Genomes for Energy and the EnvironmentGoals identify the protein machines that carry out critical life functions characterize the gene regulatory networks that control these machines characterize the functional repertoire of complex micro

43、bial communities in their natural environments develop the computational capabilities to integrate and understand these data and begin to model complex biological systemsHapMapAn NIH program to chart genetic variation within the human genome Begun in 2002, the project is a 3-year effort to construct

44、 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 id

45、entifying the DNA regions associated with common complex diseases Map may also be useful in understanding how genetic variation contributes to responses in environmental factorsDideoxy SequencingDideoxy sequencing (also called chain- termination or Sanger method) uses an enzymatic procedure to synth

46、esize 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 containsa DNA template, a primer sequence, and a DNA polymerase to initiate synthesis of a new str

47、and 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 terminate

48、s the growing chain wherever it is incorporated. Tube A has didATP, tube C has didCTP, etc./sci/techresources/Human_Genome/publicat/primer/fig12.htmlDideoxy SequencingFor 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 fragm

49、ents 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 fragm

50、ents moving faster and appearing at the bottom of the gel. Sequence is read from bottom to top/sci/techresources/Human_Genome/publicat/primer/fig12.htmlDideoxy SequencingPolyacrylamide gel with small pores is used tofractionate single-stranded DNA. In the sizerange 10 to 500 nucleotides, DNA molecul

51、esthat differ in size by only a single nucleotidecan be separatedLane 1- partial replicas terminating in GLane 2- partial replicas terminating in ALane 3- partial replicas terminating in TLane 4- partial replicas terminating in CAlberts et al. Molecular Biology of the Cell.Brown. Genomes 2Capillary

52、ElectrophoresisTechnique combines the use of gel-filled 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

53、 down by replacing the slab with hundreds of tiny gel-filled capillaries, about 100 microns (four thousandths of an inch) in internal diameter, that can be bundled into a single array for automated detection./Science-Articles/Archive/automated-DNA-sequencing.htmlCapillary ElectrophoresisThe dideoxy

54、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 an

55、alysed in a single lane of a sequencing gel. A four-colour fluorescence detector monitors the DNA as it migrates to the bottom of the gel. The fluorescence signature is used to identify the terminal nucleotide. /Science-Articles/Archive/automated-DNA-sequencing.htmlCapillary ElectrophoresisA method

56、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

57、 long time to run and have a limited reproducibility. An automated method was much more desirable, but the automation of gels requires complex robotic handling. The use of capillaries allows much higher electrical fields to be used making the separations faster. Flexible capillaries are also easily

58、incorporated into an automated instrument making sequencing cheap, fast and efficient.However a single capillary is still a bottleneck in the sequencing process. A gel is easily capable of running up to 96 samples simultaneously. To overcome this a instrument fitted with an array of capillaries was

59、developed. In the first instruments the detector moved across the array, but the time lag means some information can be missed. Now all the capillaries are simultaneously monitored using an array of photodiodes./exemplarchem/entries/2003/leeds_chromatography/chromatography/cge.htmCapillary Electroph

60、oresishttp:/elchem.kaist.ac.kr/BK21_SMS.web/2001_instanal/FIG/20011029/0003_ANG_2000_04463_DNA_CAE.pdfCapillary ElectrophoresisCapillary holds a sieving medium, which allows separation of DNA fragments based on their sizeSample is injected into the capillary by placing the end of the capillary in th