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DNA RECOMBINANT TECHNOLOGY
Agustina Setiawati
Fakultas Farmasi USD
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REFERENCEREFERENCE
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DEFINISIDEFINISI
� DNA Cloning – the act of making many identical copies of a particular piece of DNA (often a gene)
� As you know, the first stop often involves joining a piece of DNA of interest to a cloning vector using DNA ligase
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VECTORVECTOR--PlasmidPlasmid
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Plasmid is extrachromosome DNA with special characteristic:
1. F plasmid2. R plasmid3. Col plasmid4. Virulence plasmid5. Degradative plasmid
Does eucaryote have plasmid?
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VECTORVECTOR-- PlasmidPlasmid
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Plasmid to be a Cloning VectorPlasmid to be a Cloning Vector
� Small
� Have special marker
� Only one restriction site for several endonuclease enzyme
� Has promoter and RBS
� Couldn’t be transferred to one or more species
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Plasmid brings antibiotic Plasmid brings antibiotic resistance generesistance gene
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VEKTORVEKTOR-- BakteriofageBakteriofage
Main structure of phage
Infection lysis-cycle of Phage
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� Lysogenic cycle
Plasmid as Vector: pUC8 & pBR322Plasmid as Vector: pUC8 & pBR322
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Cloning Cloning PrincipalPrincipal
CLONING STEPCLONING STEP
1. Prepare DNA target and spesific vector
2. Cut DNA target and vector using retriction endonuclease
3. Ligate them
4. Transform into living cells
5. Selection
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1. CUT DNA and Vector using 1. CUT DNA and Vector using endonucleaseendonuclease
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PEMOTONGAN HpaI
1. BLUNT ENDS
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PEMOTONGANEcoRI
2. Sticky Ends
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2. 2. Ligation Ligation DNA DNA target & vectortarget & vector
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Blunt ends need higher DNA concentration to ligate one another
Sticky ends has higher efficiency due to “catch hold” each other
How to increase ligation efficiency of blunt ends?
Strategy: 1. put sticky end into Strategy: 1. put sticky end into blunt endblunt end
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Weakness?
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Strategy: 2. Put an adaptorStrategy: 2. Put an adaptor
An adaptor has sticky ends linker and could ligate one to another
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4. Transformation in Living Cells4. Transformation in Living Cells
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Transformation in animal & plant cells
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Physical Method to transform rec Physical Method to transform rec DNADNA
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5. SELECTION-PLASMID pUC8 VECTOR
Genetic mapping of pUC8
Reaction in E.coli having pUC8
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IPTG: isopropyl thiogalactoside
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5. SELECTION-PLASMID pBr322 VECTOR
Genetic mapping of pBr322
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Selection
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Binary Cloning systemBinary Cloning system
E.Coli plasmid has homology in Ti-plasmid so they can be integrated
They transformed to A.tumefacien that able to infect plant cells
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Transformation recombinant plasmid in plant
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SUCCESS SUCCESS PROBABILTY
� Calculated by CLARCK & CARBON equation
P: probability of success (95% or 99%)
a : inserted genom
b : total genom in species
Problem?
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