分子遗传学理论与技术基础ChapterIV.DNArecombinanttechnologyPPT课件

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1、IV.DNA recombinant technology1.conceptDNA recombinant.Gene clone or molecular clone.Gene engineering.*That means to cut,modify and rejoin thedifferent DNA molecules with tool enzyme,to reform a new recon molecular,the recon transfer target gene fragment into the recipient(host)cell to clonal amplify

2、,so,we can get muchOf target gene copies,that is called gene cloning.if the target gene can express in host cell in order to reform the host cells trait,that is called gene engineering.Above technology process is also called a community name-DNA recombinant.2.Technology steps of DNA recombinant.鄂p34

3、1,p348图15-5.(1)to isolate the target gene DNA fragment and the vector DNA,then to modify with same tool enzyme.(2)ligase reaction ligate the target gene DNA and vector DNA into recon.(3)the recon transformate the host cell.there is only one recon in one host cell.(4)the transformated host cells are

4、cultured in plate medium,then to screen the target gene cell clone for clone amplify.3.The key knowledge points:1)Tool Enzyme.鄂P299.concept:there are many types enzymes,Their function are different,that are got from bionts cells(much from bacteria).These enzymes work as useful tools in gene operatio

5、n experiment,so,be called tool enzyme.*types and function of tool enzyme.(1)Restriction Endonuclease(RE).*RE can identify a specific base sequence of dsDNA molecular,then to hydrolysis the phosphodiester bond of dsDNA in endodigetion way.*the types of RE:Type I specificity unstrength.Type IIIType II

6、 digetion point is specific,very useful.there are near 100 kinds.鄂p304表。(2)DNA polymerase。鄂p305.DNA polymerase can catalysis the nucleo-tides(dNTP)polymerazatioin obey theDNA template strand to synthesis a new DNA strand.There are different types of DNA polymerase,that have different function.For ex

7、ample:E.coli DNA polymerase I.Taq DNA polymerase.(3)Ligase 鄂p306.Ligase can catalysis the ligation（连接反应）of DNAs or RNAs nick.that divided to DNA ligase and RNA ligase.for example,the function of T4 DNA ligase are to ligate:*1single strand nick of dsDNA.2cohesive end of two dsDNA.3blunt end of two ds

8、DNA.4single strand nick of DNA/RNA hybrid molecular.(4)Reverse trascriptase(RTase)鄂p306.RTase also can be called RNA dependentDNA polymerase,to catalysis cDNA strand polymeraztion obey mRNA template.mRNA 5 AAAAAA3 cDNA 3 TTT-TTT5(5)Methylase(甲基化酶)鄂p307The methylase act at a specific DNA sequence of

9、some RE identified to catalysis DNA be methylated at the specific site.The methylated DNA can defense to be digested by relative RE.For example:the EcoR I.Methylase act at the EcoR I.RE identify sequences specifically,to catalysisDNA be methylated at the specific site.5-GAA*TCC-3 CH3 (鄂p308表14-6)(6)

10、Exonuclease 鄂p308The exonuclease can catabolic(降解)the terminal nucleotide of DNA molecular selectively.5 3 3 5 5 3 3 5(7)RNase.RNase can catalysis the RNA molecular catabolicing.*RNase A type:under higher density,it can cut any point in RNA molecular.but,under lower density,it can only cut c and u p

11、oints.*RNase T1 type:be used to RNA sequencing and to delete RNA mixture in DNA sample.*RNase H type:be used to catabolic RNA strand of RNA/DNA hybrid molecules.(8)DNase I(现代分子生物学手册p169)DNase can catalysis DNA molecular catabolicing.If we control the reaction density and the reaction time,the nick f

12、requency of DNA molecular can be controlled.2)How to get the target gene fragments?(1)to screen the target gene cell clone from genome library with probe,then toClone amplify.(2)to isolate genome DNA-southern blotting hybridization-to take the target gene fragments from blotting film.(3)to screen th

13、e target cDNA cell clone from cDNA library,then to clone amplify.(4)to amplify target gene fragments with PCR technology.3)Vector DNA 鄂P309.concept:vector DNA is a tool DNA mole-cular,that can carry exogenous DNA into the host cell,then to self duplication in some way.character of vector DNA:(1)that

14、 is a small DNA fragment(310 kb),and can duplicate in host cell.(2)there are several single restriction point,that is out of replicon(复制子)region for exogenous DNA insertion.(3)after exogenous DNA insertion,the vec-tor duplication cant be influenced.(4)there are selective markers for screen the posit

15、ive cell clone.For example:the Apr and the Tet resistance gene.(5)there is a regulate region to promote the target gene expression.*鄂P311图。*for example:pBR 322.a plasmid vector.the types of vector:expression vector.clone vector.insertion vector.replacement vector.*usual applicable vectors:plasmid DN

16、A.(to carry 100 kb)BAC (300 kb)YAC (2Mb)4)Joining reaction of the targetDNA and the vector DNA.鄂p346.principle:after RE cut and modify,the target DNA fragment and the vector DNA are joined together into the recon(重组子)by some way.result evaluate:(1)high recombinant frequency.(2)the recon can be cut o

17、pen again easily.(3)the recon transfer into host cell easily,and can duplicate or express.technology methods:(1)after the double enzymolysis reaction,the recon can be joined directionally.such as Bam HI and Hind III double enzymolysis reaction.*(2)artificial linker(人工连接子)method.*鄂p351.5)Recon transf

18、ormation.concept:Transformation(转化):the recons,that are made by the plasmid vector,are led into host cell.Transfection(转染):the recon,that are Trasduction(转导):made by phage DNA vector,get into host cell.*recipient cell=host cell.preparation of sensitized recipient cell:after the treatment of some tec

19、hnology methods,the recipient cell absorb the exogenous DNA(recon DNA)more easy,this process are called sensitized recipient cell.(致敏受体细胞)金冬雁p49.6)To identify the target recon cell clone and clonal amplifycation.鄂p354-355.*probe hybridization on the cultured medium plate to identify the target recon

20、 cell clone,then to pick up the target cell clone for clonal amplifying.4.Gene library.鄂p357.1)the types of gene library:(information volume).*genome DNA library.(the information of whole genome.)*chromosome specific DNA library.(the information of some one chromosome.)*cDNA library.(the information

21、 of all expressed RNA in some target tissue cells.)2)Genome DNA library.concept the DNA clone cell population are constructed by DNA recombinant tech-nology,when the DNA clone cells popula-tion include all DNA fragments of some bionts genome DNA,this genetic information bank is called genome DNA lib

22、rary.technology steps of building gene library:(1)to isolate genome DNA from tissue cells.(2)to digest genome DNA by RE.(3)the restrictional DNA fragments recom-binant with the vector DNA into recon.(4)protein enveloped the recons,then to transfect host cells.So,we can get the recons cloned bacteria

23、 cells population that include all genomes DNA fragments.this cells population is called genome DNA library.(5)library amplification.(6)probe hybridization to screen the target recon cell clone for clonal amplification.3)cDNA library.鄂 p368.cDNA:cDNA(complementary DNA areSynthesized obey the RNA mol

24、ecular as template.The reaction are catalyzed by RTase.cDNA library:to isolate total RNA from target tissue cells,to synthesis cDNA obey total RNA as template,so,the cDNA produc-tion are mix production.then to make recon clone cells population of mix cDNA fragments by DNA recombinant technology.when

25、 the cDNA recon clone cells popula-tion include all of the expressed RNAs information in some target tissue cells,that is called cDNA library of some tissue cell.that is also the RNAs information library of some tissue cells.technology steps:*(1)to isolate total RNA from target tissue.(2)to synthesi

26、s the cDNA first strand by RTase reaction,obey total RNA as template.(3)to remove the RNA template strand with alkali treatment method.(4)to synthesis cDNA second strand by DNA polymerase I reaction,obey the cDNA first strand as template.(5)to modify the double strand cDNA.(6)to make cDNA recon clone cells popu-lation by DNA recombinant technology.-(鄂 p369 图)