PRINCIP IPLE OF RECOMBINANT DNA TECHNOLOGY DEBBIE S. RETNONINGRUM SCHOOL OF PHARMACY INSTITUT TEKNOLOGI BANDUNG Recombinant DNA Technology 1
REFERENCES 1. Glick, BR and JJ Pasternak, 2003, Molecular Biotechnology: Principles and Applications of Recombinant DNA, pages 47-89; 101-110 2. Groves MJ, 2006, Pharmaceutical Biotechnology, pages 44-55 3. Brown TA, 2006, Gene Cloning & DNA analysis, pages 3-6; 8-12; 54-80; 87-97 Recombinant DNA Technology 2
RECOMBINANT DNA TECHNOLOGY Recombination of DNA molecules from different organisms: common phenomena in nature. Corynebacterium diphtheriae infected by β virus produced toxin responsible for diphtheriae symptoms. Genetic changes in this bacterium caused by virus is a genetic engineering occurs in nature. Recombinant DNA Technology 3
RECOMBINANT DNA TECHNOLOGY Duplication of this natural phenomena in laboratory Development of methods of introduction of genetic information into a organism Genetic manipulation: Escherichia coli and Bacillus subtilis (bacterium) Saccharomyces cerevisiae (yeast) Production of high-cost materials or impossible to produce by traditional means Recombinant DNA Technology 4
EXAMPLES OF RECOMBINANT PRODUCTS Recombinant antibodies Therapeutic PROTEINs: insulin, interferon, human serum albumin, human growth hormone, tissue plasminogen activator, antithrombin, blood clotting factors, limphokine, tumor necrosis factor, superoxide dismutase, and human gonadotropin Vaccines: hepatitis B, herpes, influenza, malaria Dna vaccines Gene therapy Recombinant DNA Technology 5
EXAMPLES OF RECOMBINANT PRODUCTS Agriculture Products: Transgenic Plants (Disease Resistant Plants, Pepticide Resistant Plants, Bioinsectide) Bioremediation products: degradation of fat/oil, removal of herbicides, degradation of herbicides Recombinant DNA Technology 6
RECOMBINANT DNA TECHNOLOGY (GENE CLONING) PREPARATION OF DNA: INSERT AND VECTOR LIGATION: LIGATE INSERT DNA AND VECTOR TRANSFORMATION: INTRODUCTION OF RECOMBINANT VECTOR INTO HOST CELL SELECTION: TRANSFORMANTS CONTAINING RECOMBINANT VECTOR DETECTION: THE PRESENCE OF INSERT DNA CONFIRMATION OF INSERT DNA (SIZE AND NUCLEOTIDE SEQUENCES) 1972 Stanley Cohen and Herbert Boyer Recombinant DNA Technology 7
Three approaches: GENE CLONING DNA library: genomic dna PCR products mrna cdna (eukaryotic genes contain introns) Recombinant DNA Technology 8
RECOMBINAN DNA TECHNOLOGY (GENE CLONING) C Chromosomal DNA plasmid DNA Cleavage by restriction enzyme ligation Recombinant DNA transformant Introduction into host cell 1 cell forms 1 colony clone Recombinant DNA Technology 9
Recombinant DNA Technology 10
Genomic library Collection of clones which includes complete genetic information of an organism Examples of genome size: Bacillus anthracis: 5.1 5.2 Megabases Escherichia coli: 4 Megabases Pseudomonas putida: 6.0 6.1 Megabases Staphylococcus aureus: 2.9 Megabases Recombinant DNA Technology 11
Chromosome vs Plasmid Recombinant DNA Technology 12
PLASMID Double stranded DNA Circular DNA High copy number Small size (easy to be manipulated) Replicates independently in cell Can be inserted by foreign DNA (insert DNA) Has two markers: To detect the presence of vector in host cell To detect the presence of insert DNA Recombinant DNA Technology 13
PLASMID MAP Recombinant DNA Technology 14
RESTRICTION ENZYMES To restrict viral growth in virus-infected bacteria Type II is used for recombinant DNA Cleaves phosphodiesterase bond Recognizes and cleaves palindrome Examples of palindromic sequence: 5 GAATTC 3 3 CTTAAG 5 Recombinant DNA Technology 15
RESTRICTION ENZYMES AND LIGASE DNA (restriction enzyme) DNA (ligase) 5 - NNNGAATTCNNN -3 3 - NNNCTTAAGNNN -5 EcoRI 5 - NNNG OH P AATTCNNN -3 3 - NNNCTTAA P OH GNNN -5 5 - NNNG OH P AATTCNNN -3 3 - NNNCTTAA P OH GNNN -5 ligase 5 - NNNGAATTCNNN -3 3 - NNNCTTAAGNNN -5 Recombinant DNA Technology 16
Examples of restriction enzyme EcoRI (Escherichia coli galur R): G AATTC (sticky end) BamHI (Bacillus amyloliquefaciens): G GATCC (sticky end) BglII (Bacillus globigii): A GATCT (sticky end) SalI (Streptomyces albus): G TCGAC (sticky end) PstI (Providencia stuartii 164): CTGCA G (sticky end) HindIII (Haemophilus influenzae): A AGCTT (sticky end) KpnI (Klebsiella pneumonia): GGTAC C (sticky end) XbaI (Xanthomonas bradii): T CTAGA (sticky end) HaeIII (Hemophilus aegyptius): GG CC (blunt end) Recombinant DNA Technology 17
Cloning of PCR product PCR Ligation Streptococcus pyogenes M12 Ska gene Recombinant pgemt SDM Transformation E. coli XL-Blue pgemt/ska Recombinant DNA Technology 18
LIGATION OF PCR PRODUCT INTO pgem-t A A + ligation Transformation ska GENE pgem-t recombinant pgem-t E. coli JM109 Streptococcus pyogenes Control LB/Amp Control + LB/Amp Recombinant E. coli LB/Amp/X-Gal/IPTG Recombinant DNA Technology 19
Ligation and Transformation Recombinant DNA Technology 20
Transformation Competent cell Transformation Blue-white screening Recombinant DNA Technology 21
PLASMID MAP Recombinant DNA Technology 22
Blue-white screening To determine whether foreign DNA has been inserted? In MCS (multiple cloning site), there is lacz gene (encoding for galactosidase) X-GAL blue Insert DNA will disrupt lacz gene GALACTOSIDASE is not produced X-GAL is not converted WHITE Recombinant DNA Technology 23
VECTOR TORS CLONING VECTOR ONLY TO OBTAIN DNA FRAGMENT EXPRESSION VECTOR TO OBTAIN PROTEIN Recombinant DNA Technology 24
SOME EXAMPLES OF CLONING VECTORS Name Type Host cell Remarks pbr322 Plasmid E. coli Resistancy to ampicillin and tetracycline. General purpose vector puc8 Plasmid E. coli Resistancy to ampicillin, lac screening. General purpose vector pbluescript Plasmid E. coli Resistancy to ampicillin, lac screening. λgt10 Bacteriophage E. coli Cloning of cdna YEp24 Plasmid E. coli/yeast Resistancy to ampicillin. Yeast vector, shuttle vector. Recombinant DNA Technology 25
Gel ELECTROPHORESIS Agarose or polyacrylamide DNA is negatively charged (due to phosphate group): migrated from negative to positive poles Migration of DNA is dependent on their size: migration of small DNA > large DNA Visualization: ethidium bromide / Syber Green (DNA will fluorosence with UV exposure) Recombinant DNA Technology 26
AGAROSE GEL ELECTROPHORESIS Joe School Sambrook of Pharmacy danitb Bill Sugden: Pharmaceutical agarose Biotechnology-FA gel electrophoresis 4202 Recombinant for DNA DNA Technology 27
CONFIRMATION OF RECOMBINANT pgem-t Isolation pgem-t/ska (kit Qiaprep, Qiagene) 1 2 Size of plasmid without insert < size of plasmid with insert 1 = recombinant pgem-t 2 = pgem-t without insert Recombinant DNA Technology 28
CONFIRMATION OF RECOMBINANT pgem-t Isolation of recombinant plasmid 1 2 3 Restriction Analysis of recombinant 1 2pGEM-T Blue transformant White transformant Blue transformant 1419 pb 517 pb pgem-t 881 pb Insert DNA Electropherogram of plasmid isolation From blue and white transformants Electropherogram of restriction analysis using NdeI/BamHI 1. DNA Marker puc19/hinfi 2. Restriction analysis using NdeI/BamHI Recombinant DNA Technology 29
Sequencing result of insert DNA Recombinant DNA Technology 30
Sequencing result of insert DNA Recombinant DNA Technology 31