CLONING

Etty Widayanti, SSi. MBiotech.Bagian Anatomi Sub Bagian Biologi

Fak. Kedokteran Univ. YARSI

Genetic Engineering

- gene splicing, gene cloning, molecular cloning- process cutting a gene out of a DNA strand and inserting the gene into another DNA strand.Clones Genetically identical organisms or molecules

derived from a common ancestor

Cloning Plants from Single Cells

Cloning Animals

Animals were cloned more than 20 years ago

Two techniquesEmbryo splittingNuclear transfer

www.biotechnologyonline.gov.au

Cloning by nuclear transfer

don’t live as long

not carbon copies/identical

develop diseases early

very low success rate - 0.1 - 3%

Dedifferentiation/reprogramming may not be complete or accurate

Problems

Gene Cloning

All identical to starting gene - CLONES

Gene

Host

Cloning vector Recombinant DNA

Started with: few copies

GOAL: To get enough copies of the gene to manipulate

Multiply

Ended with: Many copies.

Steps in cloning a single piece of DNA

1. Appropriate restriction sites

2. Cut vector and foreign DNA with RE

3. Run on gel to separate fragments

4. Isolate specific fragment

5. Ligate with cut vector

6. Transform host bacteria Selection

7. Grow up colonies

8. Isolate plasmid DNA

9. Cut with RE to confirm presence of foreign DNA

10. Run on gel to identify recombinant plasmids

Gene Cloning

Vector cloning Restriction Endonuclease

(restriction enzymes) and ligase

Host cell Transformation

Cloning Vectors- carrier for DNA during the recombinant

DNA process.

- plasmid-piece of free-floating DNA in thecytoplasm of bacteria.

- double-stranded, circular molecules thatreplicate independently of the chromosome.

Vector: molecule of DNA which is used to carry a foreign gene into a host cell

Promoter gene - A sequence of bases in a nucleic acid

strand, that serves as a signal to start transcription.

Chromosomal DNA construct – The gene of interest.

Antibiotic resistant gene - Are used as a marker system for

transformed cells.

Marker gene – A gene that identifies which organisms have been successfully transformed.

Cloning Vectors

Endonucleases type of enzyme in DNA strand.

produced nucleic acid strand breaks interior of nucleic acid strand.

restriction endonucleases-enzyme produced by bacteria that is used in recombinant DNA.

cuts open bacterial plasmid.

ex: EcoRI, BamHI, HindIII, HindII, PstI

Gene construct engineered to plasmid with ligasees.Plasmids back to bacterium.

EcoR1 : E. coliPstI : Providencia stuartiiHindIII : Haemophilus influenzaNotI : Norcardia otitidis-

caviarum

Common Restriction Enzymes

G

CCTAG

GATCC

G

GATCC

G

G

CCTAG

Inserting foreign DNA using restriction enzymes

GATCC

G

G

CCTAG

BamHI BamHI

Ligase

Forming recombinant DNA:

ligation

Competent cell (host cell)

Definition- a cell that is capable of taking up

DNA

Transformed cell- cell with new DNA

Definition- process of introducing free DNA into bacteria.

Transformation

Methods of Transformation

Electroporation- Electrical shock makes cell membranes

permeable to DNA- The use of an electric shock to

momentarily open or disrupt cell walls

Calcium Chloride/Heat-ShockChemically-competent cells uptake DNA

after heat shock

Conjugation the contact of bacteria that involves the

exchange of DNA with a mating tube.

Other Processes

Agrobacterium Transformation

• Agrobacterium tumefacians is a bacterium that causes a

disease known as crown gall in plants.• Infects plants by transferring its genetic material into

plant cell.• Agrobacterium transformation is the most common

technique for genetically engineered plantsBallistic gene transfer

Ballistic Gene Transfer - the use of tiny DNAcoatedprojectiles as carriers. It is important to transport DNA through the walls of intended recipient

cells.Projectiles are often known as micro projectiles Ballaistic transformation is done by using a ‘gene gun’ the gene gun has been useful in creating agricultural crops.

Host cells

Why use bacteria?

Size: Bacteria are unicellular, making them easy to work with. Multicellularorganisms are more complex and every cell would need to contain the desired genetic alteration.

Reproduction: The faster a model organism reproduces, the more generations of offspring can be quickly produced.

Safety: E.coli strain HB101; K-12 does not make people sick.

Gel electrophoresis

-ve

+ve

Size separation4.0 kb

3.0 kb

2.0 kb

5.0

3.5

2.8

2.4

1.5

2.1

Log

(kb

)

Distance migrated

Gel electrophoresis system or “gel box”

UV illumination of stained DNA fragments

separated in an agarose gel by electrophoresis

Separating and purifying DNA fragments: gel electrophoresis

DNA is negatively charged, moves to the (+) pole in electric field

Ethidium bromide intercalates DNA,

fluoresces in UV light

We can insert the gene into cells – Now what?

Selecting for transformed cells and amplifying the

product

Basic Steps

Identify the transformants Isolate transformed colonies Amplify the product

Identifying transformants

Vectors containing antibiotic resistance genes can be used

Those that took up the vector will now express antibiotic resistance

Ability to metabolize substances included in media

Multiplication of the host cells by cloning

Large scale fermenters by cloning

All genetically identical because of asexual reproduction

References

Brown, T.A. 1990. Gene cloning: An introduction. Chapman & Hall, London.

Campbell, N.A., Reece, J.B. and Mitchell, L.G. 2004. Biologi. Jilid ke-3. Ed ke-5. Penerbit Erlangga, Jakarta.

Old, R.W. And Primrose, S.B. 2003. Prinsip-prinsip manipulasi gen: Pengantar rekayasa genetik. Penerbit Universitas Indonesia, Jakarta.

Watson, J.D., Tooze, J. And Kurtz, D.T. 1988. DNA rekombinan: Suatu pelajaran singkat. Terj dari Recombinant DNA, oleh Gunarso, W. Penerbit Erlangga, Jakarta.