Cloning and Vector Chapter 3 Instructor : Prof. Myoung-Dong Kim T: 6458, mdkim@kangwon.ac.kr Room...

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