生物化學(xué)：Chapter 9 DNA-based Information Technologies

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1、Chapter 9 DNA-based Information Technologies DNA cloning and DNA amplification From genes to genome From genome to proteomes New products of biotechnology Paul Berg was awarded one-half of the 1980 Nobel Prize in Chemistry, for his fundamental studies of the biochemistry of nucleic acids, with parti

2、cular regard to recombinant DNA“. Herbert Boyer and Stanley Cohen combined their efforts in biotechnology to invent a method of cloning genetically engineered molecules in foreign cells. DNA CloningDNA cloning A clone is an identical copy how to perform DNA cloning (the method: recombinant DNA techn

3、ology of genetic engineering)DNA cloning procedures Cutting DNA at precise locations, by using restriction endonucleases; Select vectors (plasmid): capable of self-replications; Joining target DNA and vector covalently (recombinant DNA) by using DNA ligase; Transform recombinant DNA to host cells; I

4、dentify host cells containing recombinant DNA; Most common used host cells: bacterium Escherichia coli (E. coli). Three types of restriction endonucleasesType I: They cleave the DNA at non-specific sites that are 1000 bp or more from the recognition sequence.Type II: They cleave the DNA within or ve

5、ry close to the recognition sequences. Type III: They cleave the DNA about 25 to 27 bp from the recognition sequence. People Richardson, Charles C.Professor DNA ligaseDNA kinaseDNA exonuclease DNA ligaseDNA kinaseRNA polymeraseCharles C. RichardsonHarvard Medical SchoolJerard HurwitzThe Memorial Slo

6、an-Kettering InstituteDiscoverers of DNA ligaseRestriction endonucleases(restriction enzymes) Found in different bacteria species ; Recognize and cleave DNA at specific sites; Type II is mostly used (no ATP is required); The recognition sequences are 4-6 bp long and palindromic;Cloning vector (plasm

7、id) circular DNA, replicate separately from host chromosome; an origin of replication: 10-20 copies per cell; antibiotics maker. (tetR, ampR) several RE sites for cloning; small size 4,362bp: easy transformation and manipulation.pBR322:pBR322 used for cloning of foreign DNA in E. coliBacteriophage c

8、loning vector About 1/3 of genome is not essential; Foreign DNA size can be up to 23 kb long;Bacterial artificial chromosomes (BACs) cloning of very long DNA fragments (100-200 kb); contains antibiotics marker and replication origin; 1-2 copy per cell; transformation method: electroporation; Host ce

9、lls: having mutations that facilitate uptaking of large DNA. Yeast artificial chromosome (YAC) yeast (S. cerevisiae) genome: 14 x 106 bp (eukaryotic cell) shuttle vector: replicate in cells of two or more species; YAC: ori, selective markers; CEN and TEL sequences (required for chromosome maintainen

10、ce) DNA library screening sequence-based method (hybridization); DNA library (a collection of DNA clones): genomic DNA library cDNA library probe (radioactive): DNA fragment (complementary sequence of the target DNA); Identification: autoradiographyProbe designed based on amino acid sequence“degener

11、ate” (兼并) probeA typical E. coli expression vector used for expression of recombinant proteins in bacteria; Promoter: instruct RNA polymerase to bind; overexpression: recombinant proteins represent 10% of total cellular proteins. Site-directed mutagenesis to study the function of protein two approac

12、hes: change a DNA fragment; point mutation;Polymerase Chain Reaction (PCR)Kary B. Mullis PCR reaction requires: primers, DNA polymerase; dNTPs; PCR can detect and amplify as little as one DNA molecule;PCR ProcessDNA amplified by PCR can be clonedAnalysis method of a cloned gene1. Southern Blot: the

13、detection of a gene of interest by probing DNA;2. Northern Blot: probe RNA on a gel with a DNA probe;3. Western Blot: probe proteins on a gel with an antibody.What is a genome ? Genome: All of the DNA for an organismHuman Genome: Nucleus: 3.2 billion base pairs packaged into chromosomes Mitochondrio

14、n: 16,600 base pairs packaged in one circular chromosomeThe human genome project strategy(shotgun method) Each contig contained STSs (sequence-tagged site) at a distance of very 100kb; Sequencing mapped BAC or YAC clones; Identify overlapping region.Sequence-tagged site (STS，序列標(biāo)簽位點(diǎn) ): a relatively s

15、hort sequence (200 to 500 bp) that has a single occurrence in the genome and whose location and base sequence are known (mapped). Contig (重疊群): a set of overlapping clonesConstruction of a cDNA library from mRNA Reverse transcriptase:synthesize DNA on an RNA or DNA template; cDNA: complementary DNAs

16、; EST: expressed sequence tag Duplex DNAs are cloned into vector. The proportions of human genome made up of various types of sequencesGenomic sequencing timelineSize of DNA molecules and genomesOrganism Number of base pairs (kb) Total length (m) Virus Polyoma, SV40 5.1 1.7 T7 39.9 13 166 55Bacteria

17、 Mycoplasma hominis 760 260 Escherichia coli 4,700 1,360Eukaryotes Yeast (S. cerevisiae) 13,500 4,600 Drosophila 165,000 56,000 Human 2,900,000 990,000 South American lungfish 102,000,000 34,770,000Saccharomyces cerevisiae 5570 5651Schizosaccharomyces pombe 4940Fruit fly 13000Nematode worm 18000Huma

18、n 30000 - 35000Organisms Number of proteinsNumber of proteins in different organisms Methods to determine a protein function Sequence similarity, or structural relationship provides information for protein function.1) If the amino acid sequences of two proteins from two different organisms are homol

19、ogous, for example, 30% identity and 55% similarity, the two proteins very possibly have similar biological function in cells. 2) If two proteins do not have similar amino acid sequences, but do share similar three dimensional structure and have known functional domains such as ATP binding and hydro

20、lysis domains. It is possible that the two proteins have similar function. In many cases, a newly identified protein does not have evident sequential or structural relationship to known proteins. We will use the following methods to determine its function. Determine when and where this protein appea

21、rs, or in which tissue, or at what stage of development. In general, particularly for these proteins whose functions are strictly regulated, proteins are produced just before their functions are required for coming biological events. 1). Biochemical reactions, or Western Blotting analysis can be use

22、d to examine the existence of a protein. 2). Proteomics (two-dimensional electrophoresis) is popular to determine existence and amount of proteins at large scale. Tags (fused to target protein) are used in protein purificationTag: a gene encoding a peptide or protein that binds to a known ligand.GSH

23、(tag)(ligand)Non-covalently bindingUsing GST tag inProtein purification Elute with high salt bufferor buffer containing free GSHGFP (green fluorescent proteins) is used in biological researchCloning of cDNA next to a gene for GFP creates a reporter construct, and the mRNA transcript is then expresse

24、d as a fusion protein. The GFP part of the protein is visible in the fluorescence microscope. The proteins expressed only in the four touch neurons.Using epitope tag to study protein-proteininteraction Epitope tag: short protein sequence that is bound by monoclonal antibody; New proteins can be iden

25、tified by mass spectrometry.Principle Yeast Two-hybrid System (study protein-protein interaction) Yeast Two-hybrid System (study protein-protein interaction) The two fusions are created in separate yeast strains, which are then mated. Selection: Only reporter gene is expressed, the yeast can survive

26、.haploidSynteny in the mouse and human genomes The genes in human chromosome 9 and mouse chromosome 2 exhibit a very high degree of homology and the same gene order.Use of comparative genomics to identify functionally related genes P1, P2, P7 refer to proteins encoded by each species; P3 and P6 may

27、be functionally related.How to study cellular expression levels of genes ?Photolithography technique for preparing a DNA microarray; nucleotide precursors are synthesized in a photoreaction.DNA microarray The DNA (PCR amplified or synthesized) is positioned on a solid surface (usually specially trea

28、ted glass slides); To examine changes in gene expression during different growth stages or other circumstances. DNA transformation methods1) Chemicals: CaCl22) Electrophoration3) Gene Gun4) Micro InjectionThe host cells must be prepared to be competent totake up foreign DNA. Brief Introduction of Bi

29、otechnology Ti (tumor-inducing) plasmid of Agrobacterium tumefaciensTransfer of DNA to plant cells by a bacterial parasiteMetabolites produced in Agrobacterium-infected plants生長(zhǎng)素細(xì)胞分裂素冠癭堿(derived from amino acid precursors) Two modified Ti plasmid a) remove T DNA; b) E. coli shuttle vector: remove T

30、DNA; bacterial ori; antibiotic-resistance element; foreign gene Only the plasmid containing foreign gene remains in plant. A two-plasmid strategy to create a recombinant plantTomato plants engineered to be resistant to insect larvae. The healthy plant expressing a toxin protein(Bacillus thuringiensi

31、s), which is toxic to the larvae but not to humans or other organisms.Glyphosate-resistant soybean plants草甘膦 (除草劑) Use of retroviral vectors in mammalian cell cloning Foreign gene is integrated into the target cell chromosome; Problem: Integration site is randomList of Products (Recombinant Proteins

32、) Generated Using Recombinant DNA TechnologyProduct (protein) Gene expressed in (host) ApplicationHuman growth hormone (HGH) Mouse mammary tumour cell line DwarfismTissue Plasminogen activator (TPA) Chinese hamster ovary (CHO) cell line 中國(guó)倉(cāng)鼠卵巢細(xì)胞 ThrombolysisErythropoietin (CHO) cell line AnaemiaBloo

33、d clotting factor VIII* (CHO) cell line HaemophiliaF Recombinant DNA technology apllied in New PharmaceuticalsInitiated 1990 Completion originally planned for 2005 Finished sequence anticipated Spring, 2003, to commemorate the 50th Anniversary of Watson and Crick publication (Nature 171: 737-738, Ap

34、ril 25, 1953); Human Genome Project (J. Craig Venter and Francis S. Collins ) While sequencing a single human genome cost $3 billionas part of the human genome project. The Solexa sequencing allows for about $ 10,000, in next 3-5 years, probably down to $1000. Then we will know our unique genetic ch

35、aracteristics, and the information is useful for doctors diagnosis.Low cost for sequencing a persons genome Acquisition Error rate Interpretation Lack of evidence-base Privacy Information storage Discrimination Abnormality vs. normal variationChanging Clinics (Medical Genetics) Human cloning ? The technology that would make it possible to create humans by cloning, converting the natural human reproduction into a manufacturing process where genetically designed babies are made in labs.核酸研究方向 DNA復(fù)制、重組和損傷修復(fù) RNA轉(zhuǎn)錄、加工與成熟 核酸修飾 核酸與蛋白質(zhì)相互作用 非編碼核酸的結(jié)構(gòu)與功能