cloning and vector chapter 3 instructor : prof. myoung-dong kim t: 6458, mdkim@kangwon.ac.kr room...
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Cloning and Vector
Chapter 3
Instructor : Prof. Myoung-Dong Kim
T: 6458, mdkim@kangwon.ac.kr
Room 411, Ag. Bld #3
Gene Cloning
Cloning - a definitionCloning - a definition From the Greek - klon, a twig An aggregate of the asexually produced progeny
of an individual;a group of replicas of all or part of a macromolecule (such as DNA or an antibody)
An individual grown from a single somatic cell of its parent & genetically identical to it
Clone: a collection of molecules or cells, all identical to an original molecule or cell
DNA CLONINGDNA CLONING
A method for identifying and purifying a
particular DNA fragment (clone) of interest
from a complex mixture of DNA fragments,
and then producing large numbers of the
fragment (clone) of interest.
Gene cloning Gene cloning
When DNA is extracted from an organism, all its genes are obtained
In gene (DNA) cloning a particular gene is copied (cloned)
Why Clone DNA?Why Clone DNA?
A particular gene can be isolated and its nucleotide sequence determined
Control sequences of DNA can be identified & analyzed
Protein/enzyme/RNA function can be investigated
Mutations can be identified, e.g. gene defects related to specific diseases
Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.
1) Chromosomal DNA1) Chromosomal DNA
2) RNA converted to cDNA2) RNA converted to cDNA
3) PCR-amplified DNA3) PCR-amplified DNA
Sources of DNA for CloningSources of DNA for Cloning
PCR-amplified DNAPCR-amplified DNA
Cloning ToolsCloning Tools
Restriction endonucleasesRestriction endonucleases LigaseLigase VectorsVectors HostHost Methods for introducing DNA into a Methods for introducing DNA into a
host cellhost cell
Cutting DNACutting DNA
Restriction endonucleases Restriction endonucleases (restriction enzymes)(restriction enzymes)• sticky endssticky ends• blunt endsblunt ends
NomenclatureNomenclature• EcoEcoRIRI• EE = genus ( = genus (EscherichiaEscherichia))• coco = species ( = species (colicoli))• R = strainR = strain• I = # of enzymeI = # of enzyme
Blunt & Sticky endsBlunt & Sticky ends
Pasting DNAPasting DNA
Complementary Complementary ends (sticky ends (sticky ends) H-bondends) H-bond
Ligase forms Ligase forms phosphodiester phosphodiester bond to seal bond to seal strands together.strands together.
Vectors
Cloning vectorsCloning vectors
Allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
1 Plasmid vectors Plasmid vectors
2 Bacteriophage vectors Bacteriophage vectors
3 Cosmids Cosmids
4 BACs & YACs BACs & YACs
Plasmid vectorsPlasmid vectors
Advantages:
• Small, easy to handle• Straightforward selection strategies• Useful for cloning small DNA fragments
(< 10kbp) Disadvantages:
• Less useful for cloning large DNA fragments
(> 10kbp)
Plasmid vectors are double-stranded, circular, self-replicating, extra-chromosomal DNA molecules.
Plasmid vectorsPlasmid vectors
Plasmids are circular DNA molecules present in the cytoplasm of the bacteria
Capable of autonomous replication
Can transfer genes from one cell to other
Act as vectors in genetic engineering.
Can also present in Yeasts
Plasmid vectorsPlasmid vectors
may encode genetic information for properties
1 Resitance to Antibiotics 2 Bacteriocins production 3 Enterotoxin production 4 Enhanced pathogen city 5 Reduced Sensitivity to mutagens 6 Degrade complex organic molecules
T.V.Rao MD
Plasmid vector for cloningPlasmid vector for cloning
1. Contains an origin of replication, allowing for
replication independent of host’s genome.
2. Contains Selective markers: Selection of cells
containing a plasmid
twin antibiotic resistance
blue-white screening
3. Contains a multiple cloning site (MCS)
4. Easy to be isolated from the host cell.
Plasmid vectorsPlasmid vectors
Bacteriophage vectors
Advantages:• Useful for cloning large DNA fragments
(10 - 23 kbp)• Inherent size selection for large inserts
Disadvantages:• Less easy to handle
vectorsvectors
Left arm:• head & tail proteins
Right arm:• DNA synthesis• regulation• host lysis
Deleted central region:• integration &
excision• regulation
BacteriophageBacteriophage
Cosmid vectorsCosmid vectors
Advantages:• Useful for cloning very large DNA
fragments (32 - 47 kbp)• Inherent size selection for large inserts• Handle like plasmids
Disadvantages:• Not easy to handle very large plasmids • (~ 50 kbp)
Combine the properties of plasmid vectors with the useful properties of the l cos site
ZAP
BACs and YACsBACs and YACs
Advantages:• Useful for cloning extremely large DNA fragments (100 - 2,000 kbp)• This is very important for genome sequencing
projects
Disadvantages:• Not easy to handle extremely large DNA
molecules
BACs : Bacterial Artificial Chromosomes
YACs : Yeast Artificial Chromosomes
BAC vectorBAC vector
oriS and oriE mediate replication
parA and parB maintain single copy number
ChloramphenicolR marker
YAC vector YAC vector
Capable of carrying inserts of 200 - 2000 kbp in yeast
telomere telomerecentromere
URA3ARS HIS3
replicationorigin
markers
largeinserts
What determines the choice vector?
insert size
vector size
restriction sites
copy number
cloning efficiency
ability to screen for inserts
what down-stream experiments do you plan?
Expression vector
Expression vector pSE420
• Polylinker: insert desired DNAPolylinker: insert desired DNA• Amp resistanceAmp resistance
• trctrc promoter promoter• lacO lacO (operator)(operator)• Shine-Dalgarno (S/D) siteShine-Dalgarno (S/D) site (ribosome binding)(ribosome binding)• T1, T2 transcription T1, T2 transcription terminatorsterminators• lacI lacI ((lac lac repressor)repressor)
growthgrowth inducer addedinducer addedcloned gene cloned gene expressed;expressed;
product producedproduct produced
• insertion of foreign DNA insertion of foreign DNA at at BamBamHI siteHI site• tet resistance gene tet resistance gene inactivatedinactivated• transformants carrying transformants carrying foreign DNA are amp foreign DNA are amp resistant but tetracycline resistant but tetracycline sensitivesensitive
transformation:transformation: transfer transfer of genetic information of genetic information via free DNAvia free DNA
Btech6
How to clone DNA
How to clone DNAHow to clone DNA
Isolation of cloning vector (bacterial plasmid) & gene-source DNA (gene of interest)
Insertion of gene-source DNA into the cloning vector using the same restriction enzyme; bind the fragmented DNA with DNA ligase
Introduction of cloning vector into cells (transformation by bacterial cells)
Cloning of cells (and foreign genes)
Identification of cell clones carrying the gene of interest
Screening of the clone
The medium in this petri dish contains the antibiotic Kanamycin
The bacteria on the right contain Kanr, a plasmid that is resistant to Kanamycin, while the one on the left has no resistance
Note the difference in growth
Blue/White Color Screening
lacZ lacZ insert
functional enzyme nonfunctional enzyme
X-gal product X-gal product
Selecting Colonies with Recombinant Plasmids
Colony hybridizationColony hybridization
DNA probe available?• part of same gene• orthologue from another
species• synthetic oligonucleotide
Bacteriophage lambda as a cloning vector
(Fig. 10.44, p. 311, Madigan et al.)
transduction:transduction: transfer of host genes from one cell to another by a virus transfer of host genes from one cell to another by a virus
Other methods for introducing DNA
Electroporation:Electroporation: the use of an electric pulse to enable cells to take up DNA the use of an electric pulse to enable cells to take up DNA• Millisecond-length pulses open small pores in cell membranesMillisecond-length pulses open small pores in cell membranes• DNA can move into/out of the cells via pores DNA can move into/out of the cells via pores
cellcell plasmidplasmid transformanttransformant
plasmid donorplasmid donor desired transformantdesired transformant
microprojectile “gun”microprojectile “gun”
Transgenic plants may be produced with binary vector system in Agrobacterium tumefaciens
(a) generalized plant cloning vector(a) generalized plant cloning vector• ends of T-DNA (red)ends of T-DNA (red)• oriori ( (E. coliE. coli), ), oriori ( (A. tumefaciensA. tumefaciens))• resistance markers (kan, spec)resistance markers (kan, spec)
(b) can clone in (b) can clone in E. coli; E. coli; transfer totransfer to A. tumefaciens A. tumefaciens by conjugationby conjugation
(c) D-Ti = engineered Ti (to remove (c) D-Ti = engineered Ti (to remove pathogenesis genes)pathogenesis genes)
(d) D-Ti will mobilize T-DNA of (d) D-Ti will mobilize T-DNA of vector → plant cells grown in vector → plant cells grown in tissue culturetissue culture
(e) whole plants can be regenerated (e) whole plants can be regenerated from recombinant cellfrom recombinant cell
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