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Chap. 4. Molecular cloning methods - Gene Cloning

Chap. 4. Molecular cloning methods - Gene Cloning. What is a CLONE ?. genotype. Monozygotic twin. Restriction endonuclease. Haemophilus influemzae Rd ; Hamilton Smith. Hin dII. GTPyPuAC CAPuPyTG. plaque. Eco RI. Palindrome (rotational symmetry). Bacterial lawn. AATTC G.

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Chap. 4. Molecular cloning methods - Gene Cloning

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  1. Chap. 4. Molecular cloning methods - Gene Cloning What is a CLONE ? genotype Monozygotic twin

  2. Restriction endonuclease Haemophilus influemzae Rd; Hamilton Smith HindII GTPyPuAC CAPuPyTG plaque EcoRI Palindrome (rotational symmetry) Bacterial lawn AATTC G 5’-GAATTC-3’ 3’-CTTAAG-5’ G CTTAA Sticky end ; protruding, cohesive, overhang Average length ; 46 = 4,096 bp

  3. 1. Restirction endonuclease 2. ligase • Use ATP for formation of phosphodiester bond 3’-OH 5’-P 3’-OH EcoRI 5’-P PstI SmaI Sticky end ; 5’-protruding, EcoRI : 3’-protruding, PstI Blunt end; SmaI Heteroschizomer ; SmaI, XmaI cf) isoschizomer

  4. Restriction-modification system Methylases methylate the recognition sites for restriction endonuclease to protect the host DNA. The methylation is conserved during replication process. -CH3 plaque Bacterial lawn

  5. Vector plasmid pBR322 pUC19 The first letter or plasmid should be noted by lower case “p”. • Dephosphorylation ; • Alkaline phosphatase • directional cloning ; • compatible vector insert 1.Replication origin 2.Selection marker ; antibiotic resistance gene 3.Multicloning sites (MCS) Ampicillin, X-gal, IPTG β-galactosidase Self-ligation Clone  cloning

  6. P P P P Phage vector • Disadvantages of plasmid • Size • transformation efficiency Plaque hybridization colony hybridization Library, screening Lytic Lysogenic Average insert size ; ~ 15 kb Homologous gene Bacterial lawn plaque Vs. colony 혼성화 탐침 자가방사법

  7. Phagemid Cosmid, YAC, BAC ; Average length ? Helper phage • Probe ; • Polynucletide probe ; homologous gene • stringency; low or high • - temp. • - denaturing agent ; • - ionic strength • 2. Oligonucleotide probe ; 17-mer, 21-mer • - degenerated primer • U G U • UGG AUG UUC AAA AAC GA • W M F K N Q

  8. cDNA library Vs. Genomic DNA library DNA : RNA hybrid Nick translation 5’3’ polymerization 5’3’ exonuclease3’ 5’ exo. Could we directly clone a specific genomic sequence or cDNA by PCR without screening ?

  9. Kary Mullis Denaturation (94 oC) Annealing (37-72 oC) Polymerization (72 oC) Taq polymerase Thermus aquaticus Thermal cycler = PCR machine template RT-PCR

  10. Mbu-1 is specifically expressed in the mouse brain RT-PCR pancreas muscle marker spleen kidney testis heart brain lung liver A

  11. Real-time RT-PCR A Delta Rn 1 5 10 15 20 25 30 Cycle Number B 1.0 0.8 0.6 0.4 Relative level 0.2 0 liver lung brain heart testis kidney spleen muscle pancreas

  12. Expression vectors pUC19 -Toxic - Inclusion body insoluble aggregates  Inducible vector Fusion protein pTrcHis Affinity chromatography cf. heme

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