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
Host

Started with: few copies

Recombinant DNA
Multiply
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 cells.

Are used as a marker system for transformed

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
PstI
HindIII
NotI

:
:
:
:

E. coli
Providencia stuartii
Haemophilus influenza
Norcardia otitidis-caviarum

Common Restriction Enzymes

Inserting foreign DNA using restriction enzymes

Ligase
BamHI

BamHI

G GATCC
CCTAG G

G GATCC

GATCC
G

CCTAG 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
to DNA
- The use of an electric shock to
momentarily open or disrupt cell walls

permeable

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
3.5
2.8
2.4
2.1
+ve

Size separation

1.5

3.0kb
Log(kb)

ve

4.0kb

2.0kb
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.