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Gene Cloning
Cloning - a Cloning - a definitiondefinition 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.
Sources of DNA for CloningSources of DNA for Cloning
1) Chromosomal DNA1) Chromosomal DNA
2) RNA converted to cDNA2) RNA converted to cDNA
3) PCR-amplified DNA3) PCR-amplified DNA
PCR-amplified DNAPCR-amplified DNA
Cloning ToolsCloning Tools
Restriction endonucleasesRestriction endonucleases LigaseLigase VectorsVectors HostHost Methods for introducing DNA Methods for introducing DNA
into a host cellinto a host cell
Cutting DNACutting DNA
Restriction Restriction endonucleases 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.
Cloning vectorsCloning vectors
allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level. 1 Plasmid vectors Plasmid vectors
2 Bacteriophage Bacteriophage
vectorsvectors
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.
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 resistanceblue-white screening
3. Contains a multiple cloning site (MCS)4. Easy to be isolated from the host cell.
A plasmid vector for cloning A plasmid vector for cloning
Plasmid vectorsPlasmid vectors
Bacteriophage Bacteriophage vectorsvectors
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 synthesisregulationhost lysis
Deleted central region: integration &
excisionregulation
Cosmid vectorsCosmid vectors
Advantages:Useful for cloning very large DNA
fragments (32 - 47 kbp) Inherent size selection for large
insertsHandle like plasmids
Disadvantages:Not easy to handle very large
plasmids (~ 50 kbp)
combine the properties of plasmid vectors with combine the properties of plasmid vectors with the useful properties of the l the useful properties of the l cos cos sitesite
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?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 pSE420expression vector pSE420
• polylinker: insert desired polylinker: insert desired DNADNA• 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 expressed;cloned gene expressed;product producedproduct produced
(Fig. 31.4, p. 1002, Madigan et al.)
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• 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
(Fig. 10-42, p. 309, Madigan et al.)
transformation:transformation: transfer transfer of genetic information of genetic information via free DNAvia free DNA
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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 cloneScreening 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
Figure 6.12
DNA probe available? part of same gene orthologue from
another species synthetic
oligonucleotide
bacteriophage lambda as a cloning vectorbacteriophage 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
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Other methods for introducing DNAOther 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”
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transgenic plants may be produced transgenic plants may be produced with binary vector system in with binary vector system in Agrobacterium tumefaciensAgrobacterium tumefaciens
(Fig. 31.11, p. 1014, Madigan et al.)
(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 transfer toto A. tumefaciens A. tumefaciens by by conjugationconjugation
(c) D-Ti = engineered Ti (to (c) D-Ti = engineered Ti (to remove pathogenesis remove pathogenesis genes)genes)
(d) D-Ti will mobilize T-DNA of (d) D-Ti will mobilize T-DNA of vector vector → plant cells grown → plant cells grown in tissue culturein tissue culture
(e) whole plants can be (e) whole plants can be regenerated from recombinant regenerated from recombinant cellcell
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