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 techniques
Embryo splitting
Nuclear transfer
Cloning by nuclear transfer

www.biotechnologyonline.gov.au
Problems

dont 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
 Gene Cloning

GOAL: To get enough copies of the gene to manipulate

Gene Cloning vector Recombinant DNA

Host Multiply

Started with: few copies Ended with: Many copies.

All identical to starting gene - CLONES

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 the

cytoplasm of bacteria.

- double-stranded, circular molecules that

replicate independently of the chromosome.

Vector:
molecule of DNA which is used to carry
a foreign gene into a host cell
 Cloning Vectors
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.
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. coli
PstI : Providencia stuartii
HindIII : Haemophilus influenza
NotI : Norcardia otitidis-caviarum
Common Restriction Enzymes
Inserting foreign DNA using restriction enzymes

Ligase

BamHI BamHI
G GATCC G GATCC
CCTAG G CCTAG G

GATCC G
G CCTAG
Forming recombinant DNA:
ligation
Competent cell (host cell)

Definition
- a cell that is capable of taking up DNA

Transformed cell
- cell with new DNA
 Transformation

Definition
- process of introducing free DNA into
bacteria.
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-Shock
Chemically-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 plants

Ballistic gene transfer

Ballistic Gene Transfer - the use of tiny DNAcoated
projectiles 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

5.0 4.0kb Size separation

ve
3.5
3.0kb
2.8

Log(kb)
2.4
2.1 2.0kb
+ve 1.5 Distancemigrated
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.