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1、Chapter 14DNA Recombination and Recombination DNA TechnologyDept. of Biochemistry and Molecular Dept. of Biochemistry and Molecular BiologyBiologyProfessorProfessor Wu Wu YaoshengYaosheng2014-062014-06Main Contents Recombinant DNA Technology DNA recombination Gene Recombination and ClinicKey PointsD
2、NA recombination manner, homologous recombinationDNA cloning, target gene, tool enzymes, RE, vectors, plasmid, host cell, E coli.Ligation of target gene and vector Method and its mechanism of screening positive clones Alpha mutual complement experimentGene diagnosis and gene therapy 3Section OneDNA
3、Recombination4DNA Recombination Manners接合作用接合作用 (conjugation)转化作用转化作用 (transformation)转导作用转导作用 (transduction)转转 座座 (transposition)同源重组同源重组 (homologous recombination)位点特异的重组位点特异的重组(site-specific recombination)5The recombination which occur between the homologous sequences called as homologous recombi
4、nation, or general recombination）。For example, the holliday model in E.coli1. Homologous recombinationIt is the basic type of DNA recombination6n nFour key steps of HollidayFour key steps of Holliday model:model:Two homologous chromosomes align mutually trimness片段重组体片段重组体(patch recombinant)拼接重组体拼接重组
5、体(splice recombinant) Formation of intermediate Holliday by one strand break of DNA and join with another strand of another DNA Formation of heterogous double strand DNA by movement of break forkIntermediate Holliday be splited, and modified, to form two double strand recombinant DNAs ：7patch recomb
6、inant （on right site）：）： 切开的链与原来断裂的是同一条链，重组切开的链与原来断裂的是同一条链，重组 体含有一段异源双链区，其两侧来自同一亲体含有一段异源双链区，其两侧来自同一亲本本DNA。splice recombinant （on left site ）：）： 切开的链并非原来断裂的链，重组体异源双链切开的链并非原来断裂的链，重组体异源双链区的两侧来自不同亲本区的两侧来自不同亲本DNA。 8 内切酶内切酶 (recBCD)DNA侵扰侵扰(recA)分支迁移分支迁移 (recA) 内切酶内切酶(recBCD) DNA 连接酶连接酶53535353535353535353
7、5353535333535335535353Holiday中间体中间体535353539Holliday中间体中间体535353535355533355553333555533335555333355553333内切酶内切酶(ruvC)内切酶内切酶(ruvC) DNA连接酶连接酶 DNA连接酶连接酶片片段段重重组组体体拼拼接接重重组组体体10 片段重组体片段重组体（patch recombinant） 拼接重组体拼接重组体（ splice recombinant）问题：问题：1、形成哪种重组体对基因重组更有效？、形成哪种重组体对基因重组更有效？ 是拼接重组体还是片段重组体？是拼接重组体还是片段
8、重组体？ 2、同源重组原理有何应用？、同源重组原理有何应用？ “gene knockout”“gene targeting”11获获07诺贝尔生理或医学奖科学家诺贝尔生理或医学奖科学家 “在涉及胚胎干细胞和哺乳动物DNA重组方面的一系列突破性发现”及 “基因打靶” 马里奥.卡佩奇 奥利弗.史密斯 马丁.埃文斯12Embryonal stem13142. Transfer and recombinant of bacteria gene(1) Conjugation (接合作用接合作用)When the cell-cell or bacteria contact with each other
9、by pili, the plasmid DNA from a single cell (bacteria) can be transferred to another cell (bacteria). This process of DNA transfer is called conjugation.15Is possible to accept plasmid such as F factor Circular small double strand DNA separated with bacterial chromosomePlasmid 16(2) Transformation (
10、转化作用转化作用)Obtained through the automatic or artificial supply of exogenous DNA, the receptor cells or cultured cells would acquire new genetic phenotype. This process is known as the transformation.17For example：when bacteriolysis, the cleavage of DNA fragments is uptaken by another bacterial.18Multi
11、ple Choices1. The integration between two specific sites of two DNA molecular sequences catalyzed by integrated enzyme is known as:A. site specific recombination B. homologous recombination C. general recombination D. random recombination E. artificial recombination19Multiple Choices2. Transformatio
12、n means: A. phage infectionB. translocation of a geneC. Uptake of foreign DNA to cause the change of the type of cell biologyD. to produce point mutation E. to cause frameshift mutation20Multiple Choices3. The recombination occurred between homologous sequences is called A. site specific recombinati
13、on B. non-site specific recombination C. general recombination D. random recombination E. artificial recombination21Section TwoRecombinant DNA Technology222324The basic steps in gene cloning(1) To get(2) To construct(3) To transport(4) To amplify(5) To find 25(1) Some basic concepts about DNA clonin
14、gGene cloningMolecular cloning Recombinant DNA technology Genetic engineering 26The basic elements for cloning1. Target genes2. Tool enzymes3. Vectors4. Host cellsThe basic requirements 27(2) Tool EnzymesNucleasescut, shorten or degrade nucleic acid moleculesLigasesjoin nucleic acid molecules togeth
15、erPolymerasemake copies of moleculesModifying enzymesremove or add chemical groupsTopoisomerasesintroduce or remove supercoils from covalently closed-circular DNA28NucleasesDegrade DNA molecules by breaking the phosphodiester bonds Exonucleases remove one of nucleotide residues at a time from the en
16、d of a DNA moleculeEndonucleases are able to break internal phosphodiester bonds within a DNA moleculeRestriction Endonuclease There are two different kinds of nucleases29Restriction EndonucleasesThe initial observation that led to the eventual discovery of restriction endonucleases (RE) was made in
17、 the early 1950sRestriction occurs because that bacterium produces restriction endonucleases that degrades the phage DNA30The discovery of these enzymes led to Nobel prizes for W. Arber, H. Smith and D. Nathans in 1978Three different classes of RE have been recognized, but the most important one is
18、RE II which is used in DNA manipulation31The Nobel Prize in Physiology or Medicine 1978Arber WArber WNathans DNathans DSmith HSmith Hfor for the the discovery discovery of of restriction restriction enzymes enzymes and and their their application application to to problems problems of of molecular m
19、olecular geneticsgenetics32The characters of RElGenerally, 46 bases are found, mostly 6 bases, a few of 810 baseslThe sequences discriminated usually are palindrome structurelTo cut the double strands of DNA at special sites and to yield two kinds of ends: blunt ends and sticky ends33Sticky ends and
20、 Blunt ends :Sticky or cohesive ends: The resulting DNA fragments have short single-stranded overhangs at each endBase pairing between them can stick the DNA molecule back together againRestriction endonucleases with different recognition sequences may produce the same sticky ends, eg: BamH I (GGATC
21、C) and Bgl II (AGATCT)345-GGTGAATTCAGC-33-CCACTTAAGTCG55-TTGCTGCAGAAG-33-AACGACGTCTTC55-sticky end (EcoR I )3-sticky end ( Pst I )5-GGTG AATTCAGC-33-CCACTTAA GTCG5+5-TTGCTGCA GAAG-33-AACG ACGTCTTC5+35Blunt end or flush end5-CCCGGG-33-GGGCCC55-CCC GGG-33-GGG CCC5+The ligation efficiency between the b
22、lunt ends is not as high as that of the sticky terminus.Sma IlMake a simple double-stranded cut in the middle of the recognition sequence36Naming of RE Escherichia coli RY13 IEcoR IThe genus name of bacteriaThe species name of bacteriaThe strain name of bacteriaThe order of the RE found in bacteriaR
23、Es are usually named after the bacterium from which they are isolated.37Ligases To repair single-stranded breaks in double-stranded DNA molecules during DNA replicationTo join two individual fragments of double-stranded DNA together38Ligase application 39DNA ligase53355353ATPADP40PolymerasesSynthesi
24、ze a new strand of DNA complementary to an existing DNA or RNA template Four types of DNA polymerase are used routinely in genetic engineering 41Polymerases DNA polymerase I: Synthesizes dsDNA by formation of a 5,3-phosphodiester bond Klenow fragment: Come from DNA polymerase I without the N-termina
25、l fragment Reverse transcriptase: Synthesizes DNA from RNA template Taq DNA polymerase: Used in the PCR, come from bacterium Thermus aquaticus42DNA modifying enzymesAlkaline phosphatase From E.coli, calf intestinal tissue Removes the phosphate group present at the 5-terminus of a DNA molecule Polynu
26、cleotide kinase From E.coli infected with T4 phageHas the reverse effect of alkaline phosphatase, adding phosphate group onto free 5-terminus43PiPiPiPiAlkaline phosphataseTo prevent the plasmid self-cyclization 44Terminal deoxynucleotidyl transferase from calf thymus tissue adds one or more deoxyrib
27、onucleotides onto the 3- terminus of a DNADNA modifying enzymesPiPiAAAAAAA45Key PointsRestriction Endonuclease, RECharacters, Functions and ApplicationTool EnzymesNucleasesLigasePolymeraseModifying enzymes Topoisomerases 46 cDNA, cDNA library Genomic DNA, Genomic library PCR products Artificial synt
28、hesis DNA fragments(3) Target DNA 47Purification of DNA from living cellsPreparation of total cell DNA (RNA)Preparation of plasmid DNAPreparation of bacteriophage DNA48Cloning Vector(4) VectorsExpression Vector49Cloning vectorCloning vector classes Plasmid DNAPhage DNAVirus DNACloning vectors are DN
29、As which can carry target genes, transfer them into the recipient cells. 50PlasmidsBasic characters of plasmidsSmall (less than 10kb), Circular, duplex molecules of DNA containing multiple cloning sitesExist at low or high copies within bacteria, but useful plasmid present in multiple copiesContain
30、selectable markers, eg: antibiotic resistance capability conferred to bacteria51PlasmidsBasic characters of plasmidsReplicate independently from bacterial cells, which possess at least an origin site of replicationA few types of plasmid are also able to replicate by inserting themselves into the bac
31、terial chromosomeMultiply within cells quite independently from bacterial chromosome5253Lac Z -galactosidase gene Multiple cloning site (MCS)Ampicillin resistance gene54Common used phagesBacteriophage A linear dsDNA approximately 49 Kb in lengthAfter infection, it can form circular structures The ph
32、age DNA is transfered into bacterial cellsBacteriophage M13A circular ssDNA, and has been used for sequencing of a cloned target DNA fragment55CosmidsBacterial Artificial Chromosome (BAC) and Yeast Artificial Chromosome (YAC)Virus are used as vectors, eg: retro-virus, adeno-virus, adenoassociated vi
33、rus, etcOther Vectors56Key Points: PlasmidContaining multiple cloning sitesVectors PlasmidBacteriophage(, M13)CosmidsBACYACVirus DNAContaining selectable markersReplicate independently 57Multiple Choices1. Molecular cloning in the field of DNA recombination technology is called asA. the establishmen
34、t of monoclonal antibodies B. the establishment of multiple antibodies C. construction of recombinant DNA D. asexual reproduction DNA E. Sexual reproduction DNA 58Multiple Choices2. The basic condition for a target gene vector isA. that it can be replicated independentlyB. that it has multiple incis
35、ionsC. that its molecular weight is largeD. that it shouldnt have genetic markersE. that it couldnt coexist with bacteria59Multiple Choices3. Which one could not be used as a cloning vector is A. plasmid DNA B. phage DNA C. bacterial genomic DNA D. adenovirus DNA E. anti-transcript virus DNA60Multip
36、le Choices4. If the following sequence is cutted by one RE, 5GGGGGGAATTCC3, it would produce A. 5 overhang ends B. 3 overhang endsC. 5 and 3overhang endsD. 5 or 3overhang endsE. blunt ends612. The basic process of DNA cloning* The preparation of target DNA* The selection and preparation of vectors*
37、The ligation of DNA fragments in vitro * Foreign DNA is transported into host cells* The screening and identifying of target DNA62(1) The preparation of target DNAIt represents whole DNA sequence of a genome To find a fragment from genomic libraryGenomic library contains a comprehensive DNA fragment
38、s from genomic DNA cut by specific RE. During the construction of a genomic library, DNA fragments and their vectors are ligated, and then introduced into recipient cells. 63 To prepare from cDNA library or cDNAExtracting total mRNAReverse transcriptionLigationIt represents the population of mRNAs c
39、oding for genes and protein expressionTransformation Proliferation Abundant Clones 64 To prepare the gene fragment with other methodsa. PCR amplificationb. To synthesize the DNA fragment by chemical method It is typically used for those of the small biologically active peptides65(2) The selection an
40、d preparation of vectorsPlasmid phage cosmid M13 phageCapacity 10 kb 22 kb 4050 kb 200 copiesKallmann syndrome (Kallmann 综合征综合征)KALIG-1 gene deficient mutation892. Biopharmaceutical PreparationFor examples:Pharmaceutical Products by Recombinant DNA Products FunctionsProducts FunctionsCoagulation fac
41、tor III Promote blood clotting Interferon Anti-viral infections and certain cancers Erythropoietin To stimulate erythrocyte generation Interleukin Activate, stimulate various types of leukocytesbFGF, EGFStimulate cell growth and differentiation Superoxide dismutase Anti-tissue injury Growth factor T
42、reatment of dwarfism Monoclonal antibodies Diagnostic tests, cancer-oriented treatment Insulin Treatment of diabetes mellitusHepatitis B vaccine Prevention of hepatitis B 903. Gene Diagnosis and Therapy(1) Gene diagnosisGenetic testing (also called DNA-based tests, gene diagnosis) is among the newes
43、t and most sophisticated of techniques used to test for genetic disorders which involves direct examination of the DNA molecule itself. Other genetic tests include biochemical tests for such gene products as enzymes and other proteins and for microscopic examination of stained or fluorescent chromos
44、omes 913. Gene Diagnosis and Therapy(2) Gene therapyGene therapy is the use of DNA as a pharmaceutical agent to treat disease. It derives its name from the idea that DNA can be used to supplement or alter genes within an individuals cells as a therapy to treat disease. The most common form of gene t
45、herapy involves using DNA that encodes a functional, therapeutic gene in order to replace a mutated gene. Other forms involve directly correcting a mutation, or using DNA that encodes a therapeutic protein drug (rather than a natural human gene) to provide treatment. 923. Gene Diagnosis and Therapy(
46、2) Gene therapySomatic cell gene therapy (体细胞基因治疗体细胞基因治疗)Germ line gene therapy (性细胞基因治疗性细胞基因治疗)Only be used in animal experiments934. Prevention of Genetic Disease (1) Prenatal diagnosis (2) Gene test of carriers (3) Presymptomatic diagnosis (4) Genetic disease susceptibility 94Summary Recombinant
47、Technology Gene recombination Gene Recombination and ClinicMultiple Choices1. Which of the followings belongs biological preparation? A. human recombination Insulin B. Natural yeast plasmid C. E coli plasmid D. E coli phage E. yeast plamid through artificial reconstruction 96Multiple Choices2. Which
48、 of the following belongs to gene diagnosis: A. to analyze the Hb content to determine whether suffer thalassanemiaB. to determine whether favism is caused through the analysis of G-6PD activitiesC. to determine hepatitis by the test of ALT acitivities D. to diagnose whether is thalassanemia by the
49、test of -globin geneE. to test uric acid for the diagnosis of gout97Questions 1.1.1.1.为什么质粒常被用作克隆载体为什么质粒常被用作克隆载体为什么质粒常被用作克隆载体为什么质粒常被用作克隆载体? ? ? ? 需具备什么特征需具备什么特征需具备什么特征需具备什么特征? ? ? ?2.2.2.2.获取目的基因的方法有哪些获取目的基因的方法有哪些获取目的基因的方法有哪些获取目的基因的方法有哪些? ? ? ?各自有何优缺点各自有何优缺点各自有何优缺点各自有何优缺点? ? ? ?3.3.3.3.为什么说为什么说为什么说为
50、什么说RERERERE是分子生物学家中的是分子生物学家中的是分子生物学家中的是分子生物学家中的“手术刀手术刀手术刀手术刀”？其？其？其？其有何重要特征？有何重要特征？有何重要特征？有何重要特征？98Questions 4.4.4.4.如果你打算克隆一个生长因子的基因如果你打算克隆一个生长因子的基因如果你打算克隆一个生长因子的基因如果你打算克隆一个生长因子的基因, , , , 你需要什你需要什你需要什你需要什么物质来进行操作么物质来进行操作么物质来进行操作么物质来进行操作? ? ? ? 为什么为什么为什么为什么? ? ? ?5.5.5.5.哪种方法最容易将目的基因与载体相连哪种方法最容易将目的基
51、因与载体相连哪种方法最容易将目的基因与载体相连哪种方法最容易将目的基因与载体相连? ? ? ? 这种方这种方这种方这种方法是否就是最佳选择法是否就是最佳选择法是否就是最佳选择法是否就是最佳选择? ? ? ?为什么为什么为什么为什么? ? ? ?6.6.6.6.如何利用表型标记来筛选阳性克隆如何利用表型标记来筛选阳性克隆如何利用表型标记来筛选阳性克隆如何利用表型标记来筛选阳性克隆? ? ? ?99Thank you100Palindrome structureTurn to 26 AGCCTAGCGGATCCCGTTACGGTCGGATCGCCTAGGGCAATGCCGGATCCCCTAGG101回文诗欣赏回文诗欣赏清代女诗人吴绛雪写的咏四季的四首回文诗，清代女诗人吴绛雪写的咏四季的四首回文诗，春夏秋冬春夏秋冬，每首仅用十个字，却是七言绝句每首仅用十个字，却是七言绝句. . 一首诗从一首诗从1010个字中回环个字中回环出来，描写四季自然特色分明，光色陆离，让人回味无穷。出来，描写四季自然特色分明，光色陆离，让人回味无穷。被世人誉为回文诗珍品。其中，被世人誉为回文诗珍品。其中，夏夏更是公认的一枝独秀。更是公认的一枝独秀。夏夏香莲碧水动风凉，水动风凉夏日长。香莲碧水动风凉，水动风凉夏日长。长日夏凉风动水，凉风动水碧莲香。长日夏凉风动水，凉风动水碧莲香。 Turn to 11 102
