RECOMBINANT DNA TECHNOLOGY 2010 - Downloaddownload.fa.itb.ac.id/filenya/Handout...

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


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


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.


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


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


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


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


GENE CLONINGGENE CLONING

• Three approaches:

– DNA library: genomic dna

– PCR products

– mRNA cDNA (eukaryotic genes contain introns)


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



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


Chromosome Chromosome vsvs PlasmidPlasmid


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


PLASMIDPLASMID MAPMAP


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’


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’


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)


CCloningloning of of PCRPCR produproductct

PCR

Streptococcus pyogenesM12

Ska gene

Ligation

Recombinant pGEMT

SDM

Transformation

pGEMT/skaE. coli XL-Blue


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


LigationLigation and Transformationand Transformation


Transformation

Competent cell

Blue-white screening

TransformationTransformation


PLASMIDPLASMID MAPMAP


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


VEVECCTORTORSS

• CLONING VECTOR ONLY TO OBTAIN DNA FRAGMENT

• EXPRESSION VECTOR TO OBTAIN PROTEIN


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.


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)


AGAROSE AGAROSE GGEL EL ELECTROPHORESISELECTROPHORESIS

Joe Sambrook dan Bill Sugden: agarose gel electrophoresis for DNA


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


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


Sequencing result of insert DNASequencing result of insert DNA


Sequencing result of insert DNASequencing result of insert DNA

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