Upload
dinhcong
View
215
Download
1
Embed Size (px)
Citation preview
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 11
PRINPRINCCIPIPLE OF RECOMBINANT LE OF RECOMBINANT DNA TECHNOLOGYDNA TECHNOLOGY
DEBBIE S. RETNONINGRUMSCHOOL OF PHARMACY
INSTITUT TEKNOLOGI BANDUNG
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 22
REFERENCESREFERENCES
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
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 33
RECOMBINANT DNA TECHNOLOGYRECOMBINANT 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.
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 44
RECOMBINANT DNA TECHNOLOGYRECOMBINANT 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
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 55
EXAMPLES OF RECOMBINANT PRODUCTSEXAMPLES 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
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 66
EXAMPLES OF RECOMBINANT PRODUCTSEXAMPLES 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
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 77
RECOMBINANT DNA TECHNOLOGYRECOMBINANT DNA TECHNOLOGY((GENE CLONINGGENE 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 andHerbert Boyer
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 88
GENE CLONINGGENE CLONING
• Three approaches:
– DNA library: genomic dna
– PCR products
– mRNA cDNA (eukaryotic genes contain introns)
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 99
RECOMBINAN RECOMBINAN DNA DNA TECHNOLOGYTECHNOLOGY((GENE CGENE CLONING)LONING)
Chromosomal DNA
Cleavage by restriction enzymeplasmidDNA
ligation
Recombinant DNA
Introduction into host cell
transformant1 cell forms 1 colony
clone
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1010
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1111
Genomic libraryGenomic 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
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1212
Chromosome Chromosome vsvs PlasmidPlasmid
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1313
PLASMIDPLASMID
• 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
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1414
PLASMIDPLASMID MAPMAP
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1515
RESTRICTION ENZYMESRESTRICTION 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’
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1616
DNA (ligase)
5’- NNNGOH PAATTCNNN -3’3’- NNNCTTAAP OHGNNN -5’
ligase
5’- NNNGAATTCNNN -3’3’- NNNCTTAAGNNN -5’
RESTRICTION ENZYMES AND RESTRICTION ENZYMES AND LIGASELIGASE
DNA (restriction enzyme)
5’- NNNGAATTCNNN -3’3’- NNNCTTAAGNNN -5’
EcoRI
5’- NNNGOH PAATTCNNN -3’3’- NNNCTTAA P OHGNNN -5’
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1717
Examples of restriction enzymeExamples 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)
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1818
CCloningloning of of PCRPCR produproductct
PCR
Streptococcus pyogenesM12
Ska gene
Ligation
Recombinant pGEMT
SDM
Transformation
pGEMT/skaE. coli XL-Blue
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 1919
LIGALIGATTIION OF PCRON OF PCR PRODUPRODUCT CT INTO INTO pGEMpGEM--TT
ska GENE
A A
pGEM-T
+
recombinantpGEM-T
ligation Transformation
E. coli JM109
Recombinant E. coliControl +Control –
LB/Amp LB/Amp/X-Gal/IPTGLB/Amp
Streptococcus pyogenes
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2020
LigationLigation and Transformationand Transformation
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2121
Transformation
Competent cell
Blue-white screening
TransformationTransformation
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2222
PLASMIDPLASMID MAPMAP
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2323
BlueBlue--white screeningwhite 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
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2424
VEVECCTORTORSS
• CLONING VECTOR ONLY TO OBTAIN DNA FRAGMENT
• EXPRESSION VECTOR TO OBTAIN PROTEIN
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2525
SOME EXAMPLES OF SOME EXAMPLES OF CLONING VECTORSCLONING 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.
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2626
Gel ELECTROPHORESISGel 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)
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2727
AGAROSE AGAROSE GGEL EL ELECTROPHORESISELECTROPHORESIS
Joe Sambrook dan Bill Sugden: agarose gel electrophoresis for DNA
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2828
CCONFIRMAONFIRMATTIION OF RECOMBINANTON OF RECOMBINANT pGEMpGEM--TT
Isolation pGEM-T/ska(kit Qiaprep, Qiagene)
1 = recombinant pGEM-T 2 = pGEM-T without insert
1 2
Size of plasmid without insert <size of plasmid with insert
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 2929
Isolation of recombinant plasmid1 2 3
White transformantBlue transformant pGEM-T
881 pbInsert DNA
1 2
1419 pb
517 pb
Electropherogram of plasmid isolation From blue and white transformants
Electropherogram of restriction analysis using NdeI/BamHI1. DNA Marker pUC19/HinfI2. Restriction analysis using NdeI/BamHI
Restriction Analysis of recombinant pGEM-T
Bluetransformant
CCONFIRMAONFIRMATTIION OF RECOMBINANTON OF RECOMBINANT pGEMpGEM--TT
School of Pharmacy ITBSchool of Pharmacy ITB Pharmaceutical BiotechnologyPharmaceutical Biotechnology--FA 4202FA 4202 Recombinant DNA Technology Recombinant DNA Technology 3030
Sequencing result of insert DNASequencing result of insert DNA