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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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