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1、Chapter 14,DNA Recombination and Recombination DNA Technology,Dept. of Biochemistry and Molecular Biology Professor Wu Yaosheng 2014-06,Main Contents,Recombinant DNA Technology,DNA recombination,Gene Recombination and Clinic,3,Key Points,DNA recombination manner, homologous recombination DNA cloning
2、, 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 experiment Gene diagnosis and gene therapy,4,Section One,DNA Recombination,5,DNA Recombination Manners,6,The recombin
3、ation which occur between the homologous sequences called as homologous recombination, or general recombination）。,For example, the holliday model in E.coli,1. Homologous recombination,It is the basic type of DNA recombination,7,Four key steps of Holliday model:,Two homologous chromosomes align mutua
4、lly trimness,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 fork,Intermediate Holliday be splited, and modified, to form two double strand recombinant DNAs ：,8,patch recombinant
5、（on right site）： 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，重组体异源双链区的两侧来自不同亲本DNA。,9,Holiday中间体,10,内切酶 (ruvC),内切酶 (ruvC),DNA 连接酶,DNA 连接酶,片段重组体,拼接重组体,11,片段重组体 （patch recombinant）,拼接重组体 （ splice recombinant）,问题：1、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？ 2、同源重组原理
6、有何应用？,“gene knockout”,“gene targeting”,12,获07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,13,Embryonal stem,14,15,2. Transfer and recombinant of bacteria gene,(1) Conjugation (接合作用),When the cell-cell or bacteria contact with each other by pili, the plasmid DNA from a
7、single cell (bacteria) can be transferred to another cell (bacteria). This process of DNA transfer is called conjugation.,16,Is possible to accept plasmid such as F factor,Circular small double strand DNA separated with bacterial chromosome,Plasmid,17,(2) Transformation (转化作用),Obtained through the a
8、utomatic or artificial supply of exogenous DNA, the receptor cells or cultured cells would acquire new genetic phenotype. This process is known as the transformation.,18,For example：when bacteriolysis, the cleavage of DNA fragments is uptaken by another bacterial.,19,Multiple Choices,1. The integrat
9、ion 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 recombination,20,Multiple Choices,2. Transformation means:,A. phage infe
10、ction B. translocation of a gene C. Uptake of foreign DNA to cause the change of the type of cell biology D. to produce point mutation E. to cause frameshift mutation,21,Multiple Choices,3. The recombination occurred between homologous sequences is called,A. site specific recombination B. non-site s
11、pecific recombination C. general recombination D. random recombination E. artificial recombination,22,Section Two,Recombinant DNA Technology,23,24,25,The basic steps in gene cloning,(1) To get,(2) To construct,(3) To transport,(4) To amplify,(5) To find,26,(1) Some basic concepts about DNA cloning,G
12、ene cloning,Molecular cloning,Recombinant DNA technology,Genetic engineering,27,The basic elements for cloning,1. Target genes,2. Tool enzymes,3. Vectors,4. Host cells,The basic requirements,28,(2) Tool Enzymes,Nucleasescut, shorten or degrade nucleic acid molecules Ligasesjoin nucleic acid molecule
13、s together Polymerasemake copies of molecules Modifying enzymesremove or add chemical groups Topoisomerasesintroduce or remove supercoils from covalently closed-circular DNA,29,Nucleases,Degrade DNA molecules by breaking the phosphodiester bonds,Exonucleases remove one of nucleotide residues at a ti
14、me from the end of a DNA molecule Endonucleases are able to break internal phosphodiester bonds within a DNA molecule,Restriction Endonuclease,There are two different kinds of nucleases,30,Restriction Endonucleases,The initial observation that led to the eventual discovery of restriction endonucleas
15、es (RE) was made in the early 1950s,Restriction occurs because that bacterium produces restriction endonucleases that degrades the phage DNA,31,The discovery of these enzymes led to Nobel prizes for W. Arber, H. Smith and D. Nathans in 1978,Three different classes of RE have been recognized, but the
16、 most important one is RE II which is used in DNA manipulation,32,The Nobel Prize in Physiology or Medicine 1978,Arber W,Nathans D,Smith H,“for the discovery of restriction enzymes and their application to problems of molecular genetics“,33,The characters of RE,Generally, 46 bases are found, mostly
17、6 bases, a few of 810 bases The sequences discriminated usually are palindrome structure To cut the double strands of DNA at special sites and to yield two kinds of ends: blunt ends and sticky ends,34,Sticky ends and Blunt ends :,Sticky or cohesive ends: The resulting DNA fragments have short single
18、-stranded overhangs at each end Base pairing between them can stick the DNA molecule back together again Restriction endonucleases with different recognition sequences may produce the same sticky ends, eg: BamH I (GGATCC) and Bgl II (AGATCT),35,5-GGTGAATTCAGC-3 3-CCACTTAAGTCG5,5-TTGCTGCAGAAG-3 3-AAC
19、GACGTCTTC5,5-sticky end (EcoR I ),3-sticky end ( Pst I ),5-GGTG AATTCAGC-3 3-CCACTTAA GTCG5,+,5-TTGCTGCA GAAG-3 3-AACG ACGTCTTC5,+,36,Blunt end or flush end,The ligation efficiency between the blunt ends is not as high as that of the sticky terminus.,Sma I,Make a simple double-stranded cut in the mi
20、ddle of the recognition sequence,37,Naming of RE,Escherichia coli RY13 I,EcoR I,The genus name of bacteria,The species name of bacteria,The strain name of bacteria,The order of the RE found in bacteria,REs are usually named after the bacterium from which they are isolated.,38,Ligases,To repair singl
21、e-stranded breaks in double-stranded DNA molecules during DNA replication,To join two individual fragments of double-stranded DNA together,39,Ligase application,40,DNA ligase,5,3,3,5,5,3,5,3,ATP,ADP,41,Polymerases,Synthesize a new strand of DNA complementary to an existing DNA or RNA template,Four t
22、ypes of DNA polymerase are used routinely in genetic engineering,42,Polymerases,DNA polymerase I: Synthesizes dsDNA by formation of a 5,3-phosphodiester bond Klenow fragment: Come from DNA polymerase I without the N-terminal fragment Reverse transcriptase: Synthesizes DNA from RNA template Taq DNA p
23、olymerase: Used in the PCR, come from bacterium Thermus aquaticus,43,DNA modifying enzymes,Alkaline phosphatase From E.coli, calf intestinal tissue Removes the phosphate group present at the 5-terminus of a DNA molecule,Polynucleotide kinase From E.coli infected with T4 phage Has the reverse effect
24、of alkaline phosphatase, adding phosphate group onto free 5-terminus,44,Pi,Pi,Pi,Pi,Alkaline phosphatase,To prevent the plasmid self-cyclization,45,Terminal deoxynucleotidyl transferase from calf thymus tissue adds one or more deoxyribonucleotides onto the 3- terminus of a DNA,DNA modifying enzymes,
25、Pi,Pi,A,A,A,A,A,A,A,46,Key Points,Restriction Endonuclease, RE Characters, Functions and Application,Tool Enzymes,Nucleases,Ligase,Polymerase,Modifying enzymes,Topoisomerases,47,cDNA, cDNA library,Genomic DNA, Genomic library,PCR products,Artificial synthesis DNA fragments,(3) Target DNA,48,Purifica
26、tion of DNA from living cells,Preparation of total cell DNA (RNA) Preparation of plasmid DNA Preparation of bacteriophage DNA,49,Cloning Vector,(4) Vectors,Expression Vector,50,Cloning vector,Cloning vectors are DNAs which can carry target genes, transfer them into the recipient cells.,51,Plasmids,B
27、asic characters of plasmids,Small (less than 10kb), Circular, duplex molecules of DNA containing multiple cloning sites Exist at low or high copies within bacteria, but useful plasmid present in multiple copies Contain selectable markers, eg: antibiotic resistance capability conferred to bacteria,52
28、,Plasmids,Basic characters of plasmids,Replicate independently from bacterial cells, which possess at least an origin site of replication,A few types of plasmid are also able to replicate by inserting themselves into the bacterial chromosome,Multiply within cells quite independently from bacterial c
29、hromosome,53,54,Lac Z -galactosidase gene,Multiple cloning site (MCS),Ampicillin resistance gene,55,Common used phages,Bacteriophage ,A linear dsDNA approximately 49 Kb in length After infection, it can form circular structures The phage DNA is transfered into bacterial cells,Bacteriophage M13,A cir
30、cular ssDNA, and has been used for sequencing of a cloned target DNA fragment,56,Cosmids,Bacterial Artificial Chromosome (BAC) and Yeast Artificial Chromosome (YAC),Virus are used as vectors, eg: retro-virus, adeno-virus, adenoassociated virus, etc,Other Vectors,57,Key Points: Plasmid,Containing mul
31、tiple cloning sites,Vectors,Plasmid,Bacteriophage(, M13),Cosmids,BAC,YAC,Virus DNA,Containing selectable markers,Replicate independently,58,Multiple Choices,1. Molecular cloning in the field of DNA recombination technology is called as,A. the establishment of monoclonal antibodies B. the establishme
32、nt of multiple antibodies C. construction of recombinant DNA D. asexual reproduction DNA E. Sexual reproduction DNA,59,Multiple Choices,2. The basic condition for a target gene vector is,A. that it can be replicated independently B. that it has multiple incisions C. that its molecular weight is larg
33、e D. that it shouldnt have genetic markers E. that it couldnt coexist with bacteria,60,Multiple Choices,3. 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 DNA,61,Multiple Choices,4. If the following se
34、quence is cutted by one RE, 5GGGGGGAATTCC3, it would produce,A. 5 overhang ends B. 3 overhang ends C. 5 and 3overhang ends D. 5 or 3overhang ends E. blunt ends,62,2. The basic process of DNA cloning,* The preparation of target DNA,* The selection and preparation of vectors,* The ligation of DNA frag
35、ments in vitro,* Foreign DNA is transported into host cells,* The screening and identifying of target DNA,63,(1) The preparation of target DNA,It represents whole DNA sequence of a genome, To find a fragment from genomic library,Genomic library contains a comprehensive DNA fragments from genomic DNA
36、 cut by specific RE. During the construction of a genomic library, DNA fragments and their vectors are ligated, and then introduced into recipient cells.,64, To prepare from cDNA library or cDNA,Extracting total mRNA,Reverse transcription,Ligation,It represents the population of mRNAs coding for gen
37、es and protein expression,Transformation,Proliferation,Abundant Clones,65, To prepare the gene fragment with other methods,a. PCR amplification b. To synthesize the DNA fragment by chemical method It is typically used for those of the small biologically active peptides,66,(2) The selection and prepa
38、ration of vectors,Plasmid phage cosmid M13 phage,Capacity 10 kb 22 kb 4050 kb 1 kb of cloning gDNA library - + + - cDNA library + + - - Subcloning + - - + Sequencing + + - + E coli expression + + - -,67,(3) Construction of Recombinant Molecules,Both purified DNA fragments and vectors are digested wi
39、th the same restriction enzyme to give complementary cohesive ends,Analyzing the result of restriction endonuclease cleavage,Separation of molecules by gel electrophoresis Visualizing DNA molecules in a gel (EB staining) Comparison with size markers,68,69,70,Bam H切割反应,T4 DNA连接酶 15C,同一限制酶切位点连接,71,不同限
40、制酶切位点（非配伍末端）的连接,配伍末端的连接情况和同一限制酶切位点连接相似。,72,平端连接,73,同聚物加尾连接,74,人工接头及其应用,75,(4) Introduction of DNA into living cells,Serves two main purposes: Allow the recombinant DNA molecules to multiple in the host cells Purify the recombinant DNA molecules,76,Transformation -The uptake of DNA by bacterial cells
41、,Preparation of competent E.coli cells 50 mM CaCl2 is traditionally used. Another alternative way is by electro perforation,Whether the uptake results in a detectable change in the cell Whether the Cells involved is bacterial, fungal, animal or plant,77,Why does it need ? Not only a restriction dige
42、st of total cell DNA produces the fragments carrying the desired gene, but also many other fragments carrying all the other genes Numerous different recombinant DNA molecules are produced A variety of recombinant clones are yielded,(5) Screening and Identification of Recombinants,78,Correct clone,A
43、clone library,79,Direct selection,an antibiotic resistance gene Marker rescue -by complementation plasmids contain sequence (lacZ) coding for N-terminal polypeptide ( fragment) of galactosidase Mutant cells contain sequence (lacZ) coding for C-terminal polypeptide ( fragment) of galactosidase,80,By
44、complementation,Based on this blue-white colony screening,Which one is your like?,81,White clone contains the recombinant, but blue clone not contain recombinant,Multiple cloning sites,The sequence coding the N end fragment of -galactosidase,Ampr,promoter,transformation,Chromosome,The sequence codin
45、g the C end fragment of galactosidase,The growth of bacteria on the culture with X-gal,The blue clone containing the pUC18,The blue clone containing the pUC18,transformation,Cleavage N end,External DNA,Cleavage N end,Recombinant pUC18,The growth of bacteria on the culture with X-gal,The white clone
46、containing the recombinant pUC18,-mutual complement screening,Ampr,82,Methods for clone identification,Colony PCR Enzyme digestion Nucleic acid hybridization DNA sequencing,83,1kb,Figure2b.The positive plasmids after cutted by EcoR I M-1Kb DNA ladder 110, positive plasmids cutted by EcoR,Figure2a. T
47、he clones of positive plasmid M-1Kb DNA ladder 110, positive plasmid,A. before cut with RE,To identify the target gene band after cut by RE,B. after cut with RE,Identification with restriction enzymes,84,Summary,Cloning significances,Cloning requirements,Cloning tool enzymes, specially RE,Cloning vectors, specially plasmid,Cloning process,85,Multiple Choices,1. Resistance selection marker of the pUC series of vectors is,A. Kanamycin B. Tetracycline C. Ampicillin D. G418 E. Chloromycetin,86,Multiple Choices,2. The screening method for target DNA