recombinant dna technology prof. elena a. carrasquillo chapter 4 molecular biotechnology lecture 4
TRANSCRIPT
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Recombinant DNA Recombinant DNA TechnologyTechnology
Prof. Elena A. CarrasquilloChapter 4
Molecular BiotechnologyLecture 4
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Major Steps in building a DNA Major Steps in building a DNA LibraryLibrary
Cloning in Plasmid VectorIn bacteriophage vectorScreening the library DNA probe Antibody probe
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Kinds of LibrariesKinds of Libraries
Genomic Library: Stores a representation of the genome (at least one copy of a gene present)
plasmid, bacteriophage, phagemid, cosmid vectorscDNA Library: Stores a representation of
the mRNAs expressed at a certain time or stage by a microorganism or organism
plasmid, bacteriophage, phagemid, cosmid vectors
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How is a Library Built:How is a Library Built:Restriction Enzyme Mechanisms: Preparation of DNAs to be joined (a)Staggered cut: leaves “sticky ends”
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How is a Library Built:How is a Library Built:
Restriction Enzyme Mechanisms: Preparation of DNAs to be joined:
(b) Blunt End
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Ligation of DNA cut with a Ligation of DNA cut with a Restriction EnzymeRestriction Enzyme
Staggered “sticky ends”
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Ligation of DNA cut with a Ligation of DNA cut with a Restriction EnzymeRestriction Enzyme
Role of T4 DNA Ligase
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Choosing the VectorChoosing the Vector
Depends on the size of DNA to be clonedIs the protein encoded by the DNA going
to be expressed in a prokariotic or eukaryotic cell?
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Restriction Enzyme MapRestriction Enzyme MapAllows ordering of DNA fragments
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Restriction Enzyme MapRestriction Enzyme Map
We can then build maps of linear and circular molecules
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Plasmid: it’s a circular DNA Plasmid: it’s a circular DNA molecule containing:molecule containing:
a multiple cloning site or MCS an antibiotic resistance gene an origin of replication pBR322 is the basis of most engineered plasmids
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Selectable Markers:Selectable Markers:
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Kinds of plasmids in the wild:Kinds of plasmids in the wild:F plasmids-transfer information from cell
to cellR plasmids-confer antibiotic resistanceDegradative plasmids-utilization of
unusual metabolitesCryptic plasmids-No apparent function
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Other characteristics:Other characteristics:Size range form less than 1 kb to more
than 500 kbOrigin of replication-allows plasmid to
replicate in the bacteriaLow-copy number: 1-4 per cellHigh copy number: 10-100 per cellIncompatibility groups: different kinds
cannot be inside the same cell
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Characteristics of an engineered Characteristics of an engineered plasmid:plasmid:
Small size (<15Kb) for optimal efficiency of transformation in bacteria
Unique restriction enzime sites for cloningOne or more selectable genetic markers
to allow for differentiation of the plasmids carrying the cloned DNA vs the religated ones.
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Plasmid: a cloning vector or Plasmid: a cloning vector or vehiclevehicle
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pUC19 another plasmid cloning pUC19 another plasmid cloning vectorvectorCharacteristics:Interruption of b-lactamase gene gives
rise to white colonies (cloned DNA) vs blue ones (empty)
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MCS: multiple cloning site of MCS: multiple cloning site of pUC19:pUC19:
-Produced by site-directed mutagenesis to alter the DNA sequence but not the protein sequence of b-lactamase-This produced new restriction enzyme sites
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Creation of a DNA Library: Partial Creation of a DNA Library: Partial DNA digestionDNA digestion
Purpose: To produce overlapping DNA fragments
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Progress of Reaction: agarose gel Progress of Reaction: agarose gel electrophoresiselectrophoresis
Vary time of digestion or amount of enzyme units
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Screening a library:I. Screening a library:I. Colony Colony hybridization or Southern Blothybridization or Southern Blot
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Preparation of DNA ProbePreparation of DNA ProbeMethod 1Random primersEnzyme: Klenow Fragment + dNTPsNon-radioactive: biotin or chemiluminescent labeled dNTPs-Radioactive:32P
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Preparation of DNA ProbePreparation of DNA ProbeMethod 2: 5’-end labelingMethod 3: 3’end labeling
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Klenow Fragment of E. coli DNA Klenow Fragment of E. coli DNA Polymerase I Enzyme ActivitiesPolymerase I Enzyme Activities
The polymerase (red) adds deoxyribonucleotides to the 3’- hydroxyl groups of the growing chains -The 5’ exonuclease (blue) removes succesive nucleotides from the 5’ phosphate ends -The 3’ exonuclease (yellow) removes succesive nucleotides from the3’ hydroxyl ends
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Screening a Genomic LibraryScreening a Genomic Library
Colony Hybridization
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Colony Hybridization:Colony Hybridization:Procedure:
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Screening a Genomic Library: Screening a Genomic Library: II.Colony ImmunoassayII.Colony ImmunoassayThe probe is an antibodyThe colonies are grown so that the recombinant protein is expressed
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Screening a Genomic Library: Screening a Genomic Library: II.Colony ImmunoassayII.Colony Immunoassay
If chemiluminescense was used, light will be emitted and captured in an X-ray film
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Screening a Genomic Library:Screening a Genomic Library:III.Functional ComplementationIII.Functional Complementation
Defective host cells (A-) are transformed with a genomic library derived from cells that are normal with respect to that function (A+) and grown in minimal media.
Those cells harboring a plasmid that corrects the defect will grow in minimal media.
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Another Type of Library:cDNA Another Type of Library:cDNA LibraryLibraryUsed to obtain
functional eukaryotic coding regions.
E. coli does not process introns.
First step: Isolate poly A+ mRNA with oligo (dT) cellulose
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cDNA Library:cDNA Library:Second Step:Synthesis of cDNA from
mRNA of specific cells
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cDNA Library:cDNA Library:Third
Step:Selecting and Cloning Full length cDNA molecules
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Genomic Library: Bacteriophage Genomic Library: Bacteriophage λλ (Lambda)(Lambda)For cloning inserts of 10-20 KbPlasmid libraries hold up to 10 kb inserts
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Genomic Library: Bacteriophage Genomic Library: Bacteriophage λλ (Lambda)Life Cycle(Lambda)Life Cycle
Lytic Cycle:Production of progeny
Lysogenic Cycle: Integration into bacterial chromosome
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Genomic Library: Bacteriophage Genomic Library: Bacteriophage λλ
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Genomic Library: Bacteriophage Genomic Library: Bacteriophage λλ
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Cosmid libraryCosmid library
Allows cloning of 45 kbDNA fragments
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BACs: Bacterial Artificial BACs: Bacterial Artificial ChromosomesChromosomesBased on P1 bacteriophage, the F plasmid
and the lacZ region of pUC plasmidsIt’s a low copy number plasmidCarries 50-300kb fragments
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BACs: Bacterial Artificial BACs: Bacterial Artificial ChromosomesChromosomes
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YACs:Yeast Artificial YACs:Yeast Artificial ChromosomesChromosomes
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Methods of Introducing Foreign Methods of Introducing Foreign DNADNATransformationElectroporationConjugation
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ElectroporationElectroporation
Application of an electrical field to cells
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Conjugation:Tripartite matingConjugation:Tripartite mating