LEKSION 3 A

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WELCOMEN

BIOTECHNOLOGY SITE

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Gene Cloning

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Cloning - a definition

From the Greek - klon, a twig

An aggregate of the asexually producedprogeny of an individual;a group ofreplicas 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 toit

Clone: a collection of molecules orcells, all identical to an original

molecule or cell

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DNA 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.

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Gene cloning

When DNA is

extracted from an

organism, all its

genes are obtained

In gene (DNA)

cloning a particular

gene is copied(cloned)

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Sources of DNA for Cloning

1) Chromosomal DNA

2) RNA converted to cDNA

3) PCR-amplified DNA

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PCR-amplified DNA

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Cloning Tools

Restriction endonucleases

Ligase

Vectors Host

Methods for introducing DNA

into a host cell

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Cutting DNA

Restriction endonucleases(restriction enzymes)

sticky ends

blunt ends

Nomenclature

EcoRI

E= genus (Escherichia)

co = species (coli) R = strain

I = # of enzyme

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Blunt & Sticky ends

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Pasting DNA

Complementary

ends (stickyends) H-bond

Ligase forms

phosphodiesterbond to sealstrands together.

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Cloning vectors

allowing theexogenous DNA to beinserted, stored, and manipulated

mainly at DNA level.

1 Plasmid vectors

2 Bacteriophage vectors

3 Cosmids

4 BACs & YACs

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Plasmid 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.

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1. Contains an origin of replication, allowing

for replication independent of hosts

genome.2. Contains Selective markers: Selection of

cells containing a plasmidtwin 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

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Plasmid vectors

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Bacteriophage vectors

Advantages:

Useful for cloning large DNA

fragments(10 - 23 kbp)

Inherent size selection for largeinserts

Disadvantages:

Less easy to handle

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vectors Left arm:

head & tailproteins

Right arm:

DNA synthesis

regulation host lysis

Deleted central

region: integration &

excision

regulation

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Cosmid vectors

Advantages:

Useful for cloning very large DNAfragments(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

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lZAP

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BACs and YACs

Advantages: Useful for cloning extremely large DNA

fragments(100 - 2,000 kbp)

This is very important for genomesequencing projects

Disadvantages: Not easy to handle extremely large DNA

molecules

BACs: Bacterial Artificial Chromosomes

YACs: Yeast Artificial Chromosomes

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BAC vector

oriS and oriEmediate

replication parA andparB

maintain singlecopy number

ChloramphenicolRmarker

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YAC vector

Capable of carrying inserts of 200 - 2000 kbpin yeast

telomere telomerecentromere

URA3ARS HIS3

replicationorigin

markers

largeinserts

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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?

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Expression vector

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expression vector pSE420

polylinker: insert desiredDNA amp resistance

trcpromoterlacO(operator)

Shine-Dalgarno (S/D) site(ribosome binding)

T1, T2 transcriptionterminators

lacI(lacrepressor)

growth inducer addedcloned gene expressed;product produced

(Fig. 31.4, p. 1002, Madigan et al.)

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insertion of foreign DNAat BamHI site tet resistance gene

inactivated transformants carryingforeign DNA are ampresistant but tetracyclinesensitive

(Fig. 10-42, p. 309, Madigan et al.)

transformation: transferof genetic informationvia free DNA

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How to clone DNA

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How to clone DNA

Isolation of cloning vector(bacterial plasmid) & gene-source DNA (gene of interest)

Insertion of gene-source DNAinto the cloning vector usingthe same restriction enzyme;bind the fragmented DNA withDNA ligase

Introduction of cloning vectorinto cells (transformation bybacterial cells)

Cloning of cells (and foreigngenes)

Identification of cell clonescarrying the gene of interest

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Screening of the clone

The medium in this petridish contains the antibiotic

Kanamycin The bacteria on the right

contain Kanr, a plasmid thatis resistant to Kanamycin,while the one on the left hasno resistance

Note the difference ingrowth

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Blue/White Color Screening

lacZ lacZ insert

functional enzyme nonfunctional enzyme

X-gal product X-gal product

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Selecting Colonies with

Recombinant Plasmids

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Colony hybridization

Figure 6.12

DNA probe available? part of same gene orthologue from another

species synthetic oligonucleotide

b t i h l bd l i t Bt h7

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bacteriophage lambda as a cloning vector


transduction: transfer of host genes from one cell to another by a virus

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Other methods for introducing DNA

electroporation: the use of an electric pulse to enable cells to take up DNA millisecond-length pulses open small pores in cell membranes DNA can move into/out of the cells via pores

cell plasmid transformant

plasmid donor desired transformant

microprojectile gun

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transgenic plants may be produced

with binary vector system inAgrobacterium tumefaciens


(a) generalized plant cloning vector ends of T-DNA (red)ori(E. coli), ori(A. tumefaciens) resistance markers (kan, spec)

(b) can clone in E. coli;transferto A. tumefaciensbyconjugation

(c) D-Ti = engineered Ti (toremove pathogenesis

genes)(d) D-Ti will mobilize T-DNA ofvector plant cells grownin tissue culture

(e) whole plants can beregenerated from recombinant

cell

Btech15

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End

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LEKSION 3 A

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