课件参考100期生化chap21-dna bination

1、Chapter 21 DNA bination and binant DNA technologyThe Department of Biochemistry and Molecular Biology, CMUHistory1973, U.S. scientists at Stanford University to build the first binant DNA molecule; In 1980, the beginning of the construction of the first binant DNA technology to produce insulin facto

2、ries; In 1997, Rowling cloned the first animal-Dolly.Genetic bination is the exchange of information between two DNA segments. By artificial means, when a gene of one species is transferred to another living organism, it is called binant DNA technology. In common parlance, this is known as genetic e

3、ngineering.Section 1DNA bination and Gene Transfer in NatureContentsHomologous binationSite-specific binationTranspositional binationConjugationTransformation Transduction1 Homologous binationHomologous bination occurs between identical or nearly identical sequences. It is also called general binati

4、on.For example, between paternal and maternal chromosome of a pair.Homologous binationDNA invading(recA)Branch migration (recA) DNA ligase535353535353535353535353535353535335535353Holliday intermediate53535353 endonuclease (recBCD) endonuclease (recBCD)53555333 DNA ligasepatch binant 55553333 Endonu

5、clease (ruvC)53555333splice binant 5555333353555333 DNA ligase Endonuclease(ruvC) bination results in either splice or patch products, depending on how RuvC cleaves the Holliday junction. Splice products are crossover products, in which there is a rearrangement of genetic material around the site of

6、 bination. Patch products, on the other hand, are non-crossover products in which there is no such rearrangement and there is only a patch of hybrid DNA in the bination product.2 Site-specific binationSite-specific bination occurs at a specific DNA sequence. The first example was found in the integr

7、ation between DNA and E. coli DNA.DNA integrationH segmentH1 flagellinH2 flagellinrepressorP2P2hixhixHinrH1rH1Site-specific bination of bacteriaPhase variation of Salmonella typhimurium flagella 3 TranspositionTransposition is the movement of specific pieces of DNA (mobile elements) in the genome.In

8、sertion sequence（IS）TransposonTransposition resembles site-specific bination being catalyzed by special enzymes.3.1 IS transposition Insertion sequences(IS) - about 750 1500bp Including: - inverted sequences：941bp - transposase gene： - repeated sequences：412bpTypes of IS transpositionDuplicative tra

9、nspositionConservative transpositionDuplicative transpositionConservative transposition3.2 Transposon transpositionTransposonInsertion sequence + another gene (usually antibiotic gene)IS Transposase geneTransposon Transposase genetet-R geneTransposons TranspositionTransposons Transposition 4.1 Conju

10、gationBacterial Conjugation has been defined as the transmission of genetic information from a donor bacterium to a recipient cell through cell-to-cell contact. Conjugation 4.2 TransformationIntroduction of an exogenous DNA into a cell, causing the cell to acquire a new phenotype.DNA fragments (usua

11、lly about 20 genes long) from a dead degraded bacterium. Nuclease enzymes then cut the DNA into fragments.Transformation DNA fragments bind to DNA binding proteins on the surface of a competent recipient bacterium.One strand is destroyed and penetrates the recipient bacterium. This DNA fragment from

12、 the donor is then exchanged for a piece of the recipients DNA by means of Rec A proteins. Some transformations Transformation experiment of Streptococcus pneumoniae 4.3 TransductionTransduction is the transfer of DNA fragments in one bacterium into another bacterium by means of the bacteriophage in

13、fection. Bacteriaphage Growthlyticpathway lysogenicpathway Section 2DNA bination TechniqueCorrelative conceptsCloneA clone is defined as a number of identical molecules, cells or individuals all derived from a common ancestor.Also named asexual multiplication.DNA CloningDNA cloning involves separati

14、ng a specific gene or segment of DNA from its larger chromosome and attaching it to a small molecule of carrier DNA, then replicating this modified DNA thousands or even millions of times.Goal of BiotechnologyObtain a specific gene. Obtain an expression product of some specific gene.Some problems in

15、 genetic engineering?1. How to get the specific gene? 2. How to transport the target gene into a recipient cell?1 Applications in enzymology Restriction endonucleases (RE) DNA polymerase reverse transcriptase DNA ligase Alkaline phosphatase Terminal transferase Taq DNA polymeraseGGATCCCCTAGGGCCTAGGA

16、TCC G+Bam H1.1 Restriction endonucleaseIt can recognize special sequences and cleave DNA at these specific base sequences.History about REWerner Arber showed that certain enzymes of bacteria restrict the enzyme of phages into host bacteria.Hamilton Smith in 1970 isolated the first restriction enzyme

17、 Hind .Daniel Nathans in 1971 for the first time applied this restriction enzyme to cut SV 40 DNA.All the three got Nobel prize in 1978.Haemophilus influenzae d strain, the third enzymeThe first alphabet-genusThe second alphabet-speciesThe third alphabet-strainHindGenusspeciesstrainorder Nomenclatur

18、e of REBacterial DNA is not broken by restriction endonuclease, because restriction site are protected by site specific methylation.Restriction-Modification System In the host cells DNA, the sequence that would be recognized by its own restriction endonuclease is protected from digestion by methylat

19、ion of the DNA, catalyzed by a specific DNA methylase. Restriction-Modification SystemClassification of REThere are three types of RE, designated I, II, and III. Type I RE cleave DNA at random sites that can be more than 1,000 base pairs (bp) from the recognition sequence.Type III RE cleave the DNA

20、about 25 bp from the recognition sequence. Both types move along the DNA in a reaction that requires the energy of ATP. Restriction SitesType II can recognize palindrome, and cleave the DNA within the recognition sequence itself. Palindrome in Greek means “to run backwards”. These are also called in

21、verted repeat sequences, which means the nucleotide sequence in 5to 3 direction is the same in both strands.GGATCCCCTAGGGTCCAGGCCTAGGATCC G+GGATCCCCTAGGHindGTCGACCAGCTGGACCTG+blunt endssticky endsblunt ends and sticky endsBam HGGATCCCCTAGGIsocaudomers are pairs of restriction enzymes that have sligh

22、tly different recognition sequences but upon cleavage generate identical termini.Bam HBg l GGATCC CCTAGG AGATCT TCTAGAGCCTAG GATCC G+ATCTAG GATCT ABam HBg lCompatible ends, can be ligated to produce DNA sequences. Different restriction enzymes can have the same recognition site - such enzymes are ca

23、lledisoschizomers.GGATCCCCTAGGGCCTAGGATCC G+Bam HGGATCCCCTAGGGCCTAGGATCC G+Bst2. VectorIn order to introduce the human gene into bacteria, at first, the gene is transferred into a carrier, known as a vector. The term ”vector” here refers to an agent that can carry a DNA fragment into a host cell for

24、 replication.Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.Including: plasmids, Bacteriophages DNA, virus DNA .Cloning vector: Allow amplification of inserted DNA segments in a host cell under their own control systems. Expression vector:

25、 Cloning vectors with the transcription and translation signals needed for the regulated expression of a cloned gene are often called expression vectors. Plasmids are reconstructed into more efficient vectors for cloning and expression. plasmidPlasmids are circular pieces of DNA that exist outside t

26、he main bacterial chromosome and carry their own genes for specialized functions. In genetic engineering, plasmids are one means used to introduce foreign genes into a bacterial cell. Extrachromosomal DNA in bacterial cytoplasmSmall, circular, double-strandSelf-replicationFeature of plasmidPlasmids

27、can be easily exchanged between living bacteria.Plasmids confer antibiotic resistance to host bacteria.Plasmids replicate independent of bacterial DNA.Plasmids contain multiple clone site, MCSPhage phage DNA:gt phages: Insertion type vectorEMBL phages: replacement type vectorM13 phage:M13mp and pUC

28、Multiple Cloning SitesOther kinds of vectorsCosmid vectors can take up still bigger fragments of DNA.Virus vectors can be used in eukaryotes.yeast artificial chromosome (YAC)bacterial artificial chromosome (BAC)Expression vectorExpression in different host organism would require different elements,

29、although they share similar requirements, for example a promoter for initiation of transcription, aribosomal binding sitefor translation initiation, and termination signals. Prokaryotes expression vectorPromoter - commonly used inducible promoters are promoters derived from lac operon and the T7 pro

30、moter. A stronger promoter; Trp/Tryptophan Operon and Tac Promoter, a hybrid collection of both the Trp and Lac Operon promoters.Ribosome Binding Site (RBS) Follows the promoter, and promotes efficient translation of the protein of interest.Translation initiation site - Shine-Dalgarno sequence enclo

31、sed in the RBS, 8 base-pairs upstream of the AUG start codon.Eukaryotes expression vectorPolyadenylation tail: Creates a polyadenylation tail at the end of the transcribed pre-mRNA that protects the mRNA from exonucleases and ensures transcriptional and translational termination: stabilizes mRNA pro

32、duction.Minimal UTR length: UTRs contain specific characteristics that may impede transcription or translation, and thus the shortest UTRs or none at all are encoded for in optimal expression vectors.Kozak sequence: Vectors should encode for a Kozak sequence in the mRNA, which assembles the ribosome

33、 for translation of the mRNA.3 binant DNA TechnologyProcess of binant DNA Technology Isolation of target geneSelection and construction of vectorsLigation of target DNA and vectorIntroduction of binant DNA into recipient cellScreening for binant plasmidsExpressing a cloned gene3.1 Isolation of targe

34、t geneChemical synthesisObtaining from genomic DNA libraryObtaining from cDNA libraryPolymerase chain reaction (PCR) Others1. Chemical synthesisonly for simple polypeptide chain whose primary structure is clear.2. Genomic DNA library 八版p411The genomic DNA library is a collection of cloned DNA fragme

35、nts representing the entire genome of a species.Process of getting genomic DNA library 3. cDNA libraryThe cDNA library is a collection of mRNA，it only contains the exons of proteins structural genes.Process of getting cDNA library4. Polymerase Chain Reaction The polymerase chain reaction (PCR) is a

36、rapid and versatile in vitro method for amplifying DNA. 八版p407PCR PCRPCR reaction systemDNA templateA pair of primersDNA polymerase (Taq)dNTPsMg2+-containing bufferProcedures of PCRDenaturing: the template DNA is denatured to e ssDNA from dsDNA by heating.Annealing: this step allows the hybridizatio

37、n of the primers with target DNA.Extension: this process is the DNA synthesis step.This cycle is repeated several times, giving an amplified product of defined length and sequence.Application of PCRSelective DNA isolationAmplification and quantification of DNAPCR in diagnosis of diseasesVariations o

38、n the basic PCR techniqueReverse Transcription PCRIn situ PCRReal-time PCR (quantitative polymerase chain reaction, qPCR)non-specific fluorescent dyes that intercalate with any double-stranded DNAsequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporte

39、r3.2 Selection and construction of vectorsA few commonly used vectors：plasmidphagecosmid yeast artificial chromosome (YAC)Vectors used in molecular cloningVector Insert (and host) Characteristics size range Plasmid Small circular DNA 5 - 10 kb (bacteria, yeast)Bacteriophage Linear viral DNA up to 20

40、 kb (bacteria)Cosmid Hybrid of plasmid up to 50 kb (bacteria) and phageYeast artificial DNA containing yeast 200 tochromosome (YAC) centromere, telomeres, 1000 kb (yeast) and origins of replication3.3 Ligation of target DNA and vectorLigation of sticky endsBam H GGATCC CCTAGGT4 DNA Ligase16CGATCC GG

41、CCTAG+Target DNAVector binant DNATarget DNA self-ligation vector self-ligationSingle digestionEco RBg l+EcoR+ Bg lEcoR+ Bg lT4 DNA ligase16C binant DNADouble digestionmake the insert directional. Artificial linkerAdding a sequence of DNA fragment, which contains the cleavage site for restriction end

42、onuclease.Terminal ligationAdding uniform polynucleotide end by terminal transferase.Ligation of blunt endsDNA Insert Ligation (sticky-end and blunt-end) into Vector DNACCGAATTCG GGCTTAAGC5-3-EcoRArtificial linker The addition of a homopolymer tail T(T)nTA(A)nAA(A)nA T(T)nT binant DNA TA cloning3.4

43、Introduction of binant DNA into recipient cellIntroduction: transformation transfection infectionRecipient cellsSafe host bacteriaEndonuclease and binase deficient Competent cells TransformationPlasmids can be introduced into bacterial cells by a process called transformation.To get the cells to tak

44、e up the DNA, the cells and DNA are incubated together at 0 in a calcium chloride solution, the subjected to heat shock by rapidly shifting the cells to temperatures of 3743. Cells so treated e”competent” to take up the DNA.Transfection: The process of DNA being introduced into eukaryotic cells.Infection: The process of virus being introduced into cells. 3.5 Screening for binantDirect SelectionAntibiotic resistanceMark