【正文】 Rebinant plasmid Inserted gene Selfligated vector: blue transformants Rebinant plasmid: containing inserted DNA, white transformants Selfligated vector (no insert) Rebinant plasmid (contain insert) Transfer to nitrocellulose or nylon membrane Denature DNA(NaOH) Bake onto membrane Probe with 32Plabled DNA plementary to gene of interest Expose to film Select positive from master plate Keep master plate (3). Colony hybridizationSouthern blot Herbert W. Boyer Professor in University of California, San Francisco Stanley Cohen Professor in Standford University Invent Rebinant DNA Technology (geic engineering) Rebined genes that originated from different anisms First experiment about Rebinant DNA technology ampR tetR 對(duì)四環(huán)素和氨芐青霉素都有抗性的新細(xì)菌菌株 Genentech pany (Geic Engineering Technology, Inc.) first biotechnology corporation, which was founded in 1976 by venture capitalist Robert A. Swanson and biochemist Dr. Herbert W. Boyer Overexpression of human insulin (胰島素） gene in Bacterial factory producing large amounts of Human proteins (insulin) 88 ? Express cloned genes encoding useful protein in different host anism ? Example: expression of human insulin in bacteria Expression vectors (表達(dá)載體 ): allowing the exogenous DNA (外源 DNA) to be stored and expressed in an anism. E. coli expression vector Yeast expression vector Mammalian expression vector Features: The origin of replication, selective marker, multiple cloning site Contain a promoter and terminator for transcription The inserted gene must have a start codon and a stop codon for translation (ORF) T7 promoter RBS Start codon MCS Transcription terminator Ampr ori T7 expression vector 原核生物常用表達(dá)載體 91 CHAPTER 8: The replication of DNA ?Molecular Biology Course 92 The Chemistry of DNA ? DNA synthesis requires deoxynucleoside triphosphates and a primer:template junction ? DNA is synthesized by extending the 3’ end of the primer ? Hydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis 93 Figure 83 Substrates required for DNA synthesis 94 Semiconservative Replication (半保留復(fù)制 ) Three hypotheses for DNA replication 95 The mechanism of DNA Polymerase (Pol) CHAPTER 8 The replication of DNA 96 Polymerase chain reaction An important technique based on DNA Polymerase The polymerase chain reaction(PCR) is to used to amplify a sequence of DNA in vitro, using a pair of primers each plementary to one end of the the DNA target sequence. 理論 技術(shù) ? Denaturation (變性 ): The target DNA (template) is separated into two stands by heating to 95℃ ? Primer annealing (退火 ): The temperature is reduced to around 55℃ to allow the primers to anneal. ? Polymerization (elongation, extension) (延伸 ): The temperature is increased to 72℃ for optimal polymerization step which uses dNTPs and requires Mg++. The principle of PCR: Three different steps proceed in each PCR cycle. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Denaturation Annealing Extension (DNA polymerase) Cycle 1 Cycle 2 Cycle 3 99 Initial DNA 8 4 2 1 Number of DNA molecules (2n): 指數(shù)擴(kuò)增 Many cycles (2535 in mon) are performed to plete one PCR reaction, which resulted in an exponential amplification of the target DNA in which both forward and reverse primers pair. 101 DNA template (模板 ) Any source of DNA that provides one or more target molecules can in principle be used as a template for PCR. Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed. 103 Figure 83 Substrates required for DNA synthesis 104 PCR Primers on opposite strands of the target sequence: forward and reverse primers （正向引物和反向引物） similar G+C contents (Tm) so that they anneal to their plementary sequences at similar temperatures (anneal temperature: Tm5176。 C) 5’ATTCCGATCGCTAATCGATGGC TCCTGTGCA TTTCGCCACTAGAG3’ 3’TAAGGCTAGCGATTAGCTACCGAGGACACGTAAAGCGGTGATCTC5’ 5’ATTCCGATCGCTAATCGATG3’ 3’CACGTAAAGCGGTGATCTC5’ forward primer reverse primer 5’CTCTAGTGGCGAAATGCAC3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Denaturation Annealing Extension (DNA polymerase) Cycle 1 Cycle 2 Cycle 3 107 PCR Enzymes ? Denaturation (變性 ): The target DNA (template) is separated into two stands by heating to 95℃ ? Primer annealing (退火 ): The temperature is reduced to around 55℃ to allow the primers to anneal. ? Polymerization (elongation, extension) (延伸 ): The temperature is increased to 72℃ for optimal polymerization step which uses dNTPs and requires Mg2+. 110 ? Taq polymerase ： isolated from the thermophilic bacterium Thermus aquaticus (嗜熱菌） , 耐熱DNA聚合酶，耐高溫 ? It has no 3’ to 5’ proofreading exonuclease activity ? Highaccuracy DNA polymerase is available mercially 111 Figure 83 Substrates required for DNA synthesis dNTP 112 PCR儀 PCR擴(kuò)增的平臺(tái)期 115 Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties Gel Electrophoresis （ 凝膠電泳） 116 and RNA molecules are negatively charged, thus move in the gel matrix (膠支持物 ) toward the positive pole (正電極 ) DNA molecules are separated according to sizes. The large DNA molecules move slower than the sm