Basics of Molecular Cloning.ppt

《Basics of Molecular Cloning.ppt》由会员分享，可在线阅读，更多相关《Basics of Molecular Cloning.ppt（21页珍藏版）》请在得力文库 - 分享文档赚钱的网站上搜索。

1、Promega CorporationEducation ResourcesUnit 006Defining Cloning“Cloning”is a loaded term that can be used to mean very different things.Cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism.(Sometimes called subcloning,because only part

2、of the organisms DNA is being cloned.)Using nuclear DNA from one organism to create a second organism with the same nuclear DNARestriction EnzymesRestriction Enzymes(also called Restriction Endonucleases)are proteins that cleave DNA molecules at specific sites,producing discrete fragments of DNA.Res

3、triction Enzymes(RE)were first isolated by Nathans and Smith in 1970.Why Restriction Enzymes?Why would bacterial cells contain proteins that cleave DNA at specific sequences?Generally restriction enzymes are thought to protect bacterial cells from phage(bacterial virus)infection.Bacterial cells that

4、 contain restriction enzymes can“cut up”invasive viral DNA without damaging their own DNA.Joining DNA FragmentsIn 1972,Paul Berg and colleagues made the first“artificial”recombinant DNA molecule.Demonstrated that the DNA of Simian virus 40 could be linearized by EcoR1Created a circular DIMER of Simi

5、an virus DNA by joining two linearized fragmentsAlso inserted pieces of Lambda phage DNA into linearized Simian 40 virus molecule.Isolating GenesHerbert Boyer and Stanley Cohen built on the work of Berg,Nathans and Smith to use restriction enzymes to isolate a single gene,place it into a plasmid vec

6、tor.Bacterial cells were then transformed with the recombinant plasmid.The bacteria host cells replicated the plasmid,producing many copies of the gene,thus amplifying it.The practical application was that expensive human protein products,like insulin,which were used to treat disease,could eventuall

7、y be produced from recombinant molecules in the laboratory using bacteria or another host.Human protein products like insulin could be used in very large quantities from the recombinant molecule.Patients no longer had to use insulin isolated from pigs or cows.Plasmid VectorsPlasmids are circular pie

8、ces of DNA found naturally in bacteria.Plasmids can carry antibiotic resistance genes,genes for receptors,toxins or other proteins.Plasmids replicate separately from the genome of the organism.Plasmids can be engineered to be useful cloning vectors.Plasmid Vectors(continued)Plasmid vectors can be de

9、signed with a variety of features:Antibiotic resistanceColorimetric“markers”Strong or weak promoters for driving expression of a proteinAntibiotic Resistance MarkersAntibiotic Resistance GeneMultiple Cloning RegionMultipleCloning RegionThe cloning marker for this plasmid is the lacZ gene.Cloning a P

10、iece of DNAAvaICut plasmid vectorwith AvaIAvaIAvaI53Excise DNA insert of interest from source using Ava ILigate the insert of interest into the cut plasmidPerforming the Restriction DigestsYou will need to set up a restriction digest of your plasmid vector and your DNA of interestRestriction enzymes

11、 all have specific conditions under which they work best.Some of the conditions that must be considered when performing restriction digest are:temperature,salt concentration,and the purity of the DNAPurify your DNA FragmentsThe insert of interest that you want to clone into your plasmid needs to be

12、separated from the other DNAYou can separate your fragment using Gel ElectrophoresisYou can purify the DNA from the gel by cutting the band out of the gel and then using a variety of techniques to separate the DNA from the gel matrixLigationLigation is the process of joining two pieces of DNA from d

13、ifferent sources together through the formation of a covalent bond.DNA ligase is the enzyme used to catalyze this reaction.DNA ligation requires ATP.Transforming BacteriaAfter you create your new plasmid construct that contains your insert of interest,you will need to insert it into a bacterial host

14、 cell so that it can be replicated.The process of introducing the foreign DNA into the bacterial cell is called transformation.Competent Host CellsNot every bacterial cell is able to take up plasmid DNA.Bacterial cells that can take up DNA from the environment are said to be competent.Can treat cell

15、s(electrical current/divalent cations)to increase the likelihood that DNA will be taken upTwo methods for transforming:heat shock and electroporationSelecting for TransformantsThe transformed bacteria cells are grown on selective media(containing antibiotic)to select for cells that took up plasmid.F

16、or blue/white selection to determine if the plasmid contains an insert,the transformants are grown on plates containing X-Gal and IPTG.(See notes for slide 11.)What did the cells take up?Plasmid onlyPlasmid with insert clonedForeign DNA from the environmentNothingExpressing your cloned geneEven if y

17、our plasmid contains insert,it may not be able to generate functional protein from your cloned DNA.The gene may not be intact,or mutations could have been introduced that disrupt it.The protein encoded by the gene may require post-translational modifications(i.e.,glycosylation or cleavage)to functio

18、n.Also,some enzymes are a complex of peptides expressed from separate genes.Expressing your cloned geneExpression of a cloned gene can be accomplished by:The E.coli hostMammalian cells(if the plasmid used is designed for expression in mammalian cells)Using an in vitro using a cell-free system.(See education resources Unit 001:The relationship between genes and proteins)