Day_2_-_DNA_manipulation

Day_2_-_DNA_manipulation - Some Basics for DNA...

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Some Basics for DNA Manipulations 1. Restriction endonucleases and DNA modification 2. Electrophoresis 3. Denaturation and hybridization 4. Chemical synthesis of oligonucleotides 2. Ligation 3. Vectors and DNA cloning 4. DNA sequencing 5. PCR, Multiplex PCR and RT PCR 6. Fluorescent in situ hybridization 7. In vitro mutagenesis
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Restriction and Restriction Endonucleases Restriction endonucleases are enzymes that cut both strands of dsDNA at very specific nucleotide sequences called restriction sites. They protect prokaryotes from infections by foreign DNAs. Why are they called “restriction enzymes”? Most T4 DNA cut at least once by a K12 restriction enzyme T4 DNA “modified” and protected from being cut by E. coliB restriction enzymes CONCLUSION: T4 phage produced in E. coli B is “restricted” to E. coli B for efficient reproduction E. coliB E. coliK12 10,000 plaques 1-5 plaques of T4(K12) Infect with 10,000 PFU ( plaque-forming units ) of T4(B) Phage T4 E. coli B many T4(B) phage progeny infect lysis
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E coli K12 E. coliB E. coliK12 1-5 plaques 10,000 plaques of T4(K12) 1-5 plaques formed by phage that escaped being cut by K12 restriction enzymes Infect with 10,000 PFU of T4(B) Infect with 10,000 PFU from plaques formed by phage that escaped destruction in E. coli K12 CONCLUSION: The phage progeny of the phage that escaped destruction in E. coli K12 has been MODIFIED for efficient growth in E. coli K12, but no longer is protected from destruction in E. coli B ! Modification and DNA Modification Enzymes
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EcoRI: --GAATTC-- --CTTAAG-- Restriction enzymes and restriction sites --G OH P AATTC-- --CTTAA PO H G-- E. coliRY13 Sau3A BamHI PstI HindIII SmaI NotI --GGATCC-- --CCTAGG-- --CTGCAG—- --GACGTC-- --AAGCTT-- --TTCGAA-- --GATC-- --CTAG-- --CCCGGG-- --GGGCCC-- --GCGGCCGC-- --CGCCGGCG-- B acillus am yloliqefaciens H H aemophilus in fluenzae R d N ocardia ot iditis P rovidencia st uartii S taphilococcus au reus 3A erratia ma rcescens What is modification ? E. coliC does not have a restriction/modification system
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N-6 methyl adenine (mA) 5-methyl cytosine (5-mC) N4-methyl cytosine (N4-mC) The three most common methylated DNA bases Modification Modification frequently involves the addition of a methyl group from S-adenosyl methionine (SAM) to C or A in a restriction site, blocking cleavage of the DNA by the corresponding restriction endonuclease. CH 3 SAM
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M S EtBr stained gels mtDNA Plasmid Apparatus Agarose-Gel Electrophoresis Ethidium bromide
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Agarose-gel electrophoresis Viewing on UV Transilluminator
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+ Direction of migration W e l l s Size of fragment is inversely proportional to its distance of migration Agarose gel electrophoresis of DNA fragments EtBr stained gel Marker DNA Eco RI Hin dIII Bam HI
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Sticky ends Digest with Bam HI Low temperature T4 DNA ligase + ATP Ligate Sticky complementary ends of restriction fragments and in vitrojoining of fragments by ligation Fig 1.26
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DNA cloning Inserted DNA has no ori but is replicated every time the vector replicates Fig 1.27
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Pst I EcoRI BamHI oriV REPLICON A primitive DNA cloning vector pBR322
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The plasmid cloning vector pUC19 Synthetic in-frame polylinker ATG Makes the plasmid a replicon
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Insertion of a piece of DNA into the plasmid cloning vector pUC19
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Day_2_-_DNA_manipulation - Some Basics for DNA...

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