12/5/2009

Kloning & Rekayasa

Genetika
Kloning
DNA rekombinan
Kloning DNA
Aplikasi teknologi
DNA rekombinan
dan DNA kloning

Bioteknologi

Biotechnology refers to technology used

to manipulate DNA. The procedures are
often referred to as genetic engineering.
DNA is the genetic material of all living
organisms and all organisms use the same
genetic code. Genes from one kind of
organism can be transcribed and translated
when put into another kind of organism.
For example, human and other genes are
routinely put into bacteria in order to
synthesize products for medical treatment
and commercial use. Human insulin, human
growth hormone, and vaccines are
produced by bacteria.
Recombinant DNA refers to DNA from two
different sources. Individuals that receive
genes from other species are transgenic.

12/5/2009

Kloning
A clone is an organism which is
genetically identical to its
parent. Clones are the result of
asexual reproduction where only
one parent is involved. The new
organism is formed by mitotic
cell division
Clones frequently occur naturally.
Potato plants reproduce
vegetatively by growing tubers
from which the new plant will
grow. Potatoes are clones.

Cloning in nature

A potato tuber is
genetically
identical to the
parent plant

12/5/2009

Other examples of cloning in nature are:

A colony of bacteria. Each bacterium splits into
two, with the total number doubling every
twenty minutes - all genetically identical.
A clump of daffodils. The new plants arising
from the original bulb are exact replicas or
clones of the parent (and of each other).
Strawberry or blackberry runners are clones of
the parent plant.

Artificial plant cloning

Cloning can also happen as a result of
human intervention. Tissue culture or
micro-propagation is a way of propagating
plants very quickly by taking a small number
of cells from a 'parent' plant and growing
them in a medium rich in nutrients and plant
growth hormones

12/5/2009

Plant cloning using tissue culture

This technique involves the following steps:

A small amount of parent tissue or a number of cells

are taken and
transferred to plates containing sterile nutrient agar
jelly,
Auxins are added to stimulate the cells to divide by
mitosis
Cells grow rapidly into small masses of tissue
More growth hormones are added to stimulate the
growth of roots and stems
The tiny plantlets are transferred into potting trays
where they develop into plants

12/5/2009

Animal cloning
Cloning also takes place naturally in the
simpler animals. Amoebas are single-celled
protozoa which reproduce by binary
fission, resulting in two offspring with identical
genes

In more complex animals, cloning occurs when
a fertilised egg splits to give identical twins.

Binary fission in the amoeba

12/5/2009

Artificial cloning of animals is now commonplace in laboratories.

The most famous example of animal cloning is Dolly the Sheep,

born in the UK in 1996 using a technique called embryo
transplanting.

Here's how it was done:

1. An egg cell was removed from the ovary of an adult female
sheep, and the nucleus removed.
2. Using micro-surgical techniques, the empty egg cell was
fused with DNA extracted from an udder cell of a donor
sheep
3. The fused cell now began to develop normally, using the
donated DNA.
4. Before the dividing cells became specialised the embryo
was implanted into the uterus of a foster-mother sheep. The
result was Dolly, genetically identical to the donor sheep.

Kloning pada
domba,
menghasilkan
domba Dolly pada
tahun 1996 di
Ingris

12/5/2009

Rekayasa Genetika

Genetic engineering (also known as genetic
manipulation or GM is not the same as
cloning. Though cloning techniques are used
in genetic engineering, the two things should
not be confused.

In the past, humans have brought about
change in the genetic make-up of organisms by
means of selective breeding. Genetic
engineering brings about such change by
scientifically altering an organism's genetic
code

Perbedaan kloning & genetic enginering

Cloning
Produces exact copies

Genetic engineering
Produces a totally unique
set of genes

Genes replicated within Genes can be swapped

the same species
across species

12/5/2009

In genetic engineering enzymes are used to

cut up and join together parts of the DNA of
one organism, and insert them into the DNA of
another organism.
In the resulting new organism the inserted
genes will code for one or more new
characteristics - for example producing a new
substance, or performing a new function. The
organism has been genetically reengineered

The diagram shows how a bacterium's genetic make-up can modified by

splicing a gene into its DNA.
This technique is also known as gene splicing or recombinant DNA
technology (because the DNA is recombined in the vector molecule).

12/5/2009

Teknologi DNA Rekombinan

Recombinant DNA is DNA that has been
created artificially. DNA from two or more
sources is incorporated into a single
recombinant molecule.

Vectors
Vectors are DNA used to transfer genes
into a host cell.
A vector must be capable of selfreplicating inside a cell.
Marker genes can be used to determine
if the gene has been taken up.
Marker genes must have some
distinguishable characteristic.
For example if you put a gene that
enables an ampicillin resistance on the
same vector as the same vector as the
gene for human insulin production, then
any bacteria that grow on an ampicillin
plate will be able to produce insulin.

12/5/2009

Plasmids
The host bacterium takes up the plasmid,
which includes the foreign gene.
When the bacteria reproduces, the
plasmids are also reproduced. The gene is
cloned.
Shuttle vectors are plasmids that are
capable of existing in several different
species. They are useful when transferring
genes to multicellular organisms.

Viruses
Viruses are the vectors of choice
for animal cells.
They can accept larger amounts of
DNA than plasmids.
When the virus reproduces within
the animal cell, it also reproduces
the foreign gene that it carries.
The gene is therefore cloned.
The DNA of some retroviruses
becomes integrated into the host
chromosome.

10

12/5/2009

Restriction enzymes
Restriction enzymes were discovered in bacteria.
Bacteria use them as a defense mechanism to
cut up the DNA of viruses or other bacteria.
Hundreds of different restriction enzymes have
been isolated. Each one cuts DNA at a specific
base sequence. For example, EcoRI always cuts
DNA at GAATTC as indicated below.

Contoh enzim restriksi

Enzyme

Cutting Site

Bam HI

GGATCC

Hae III

GGCC

Pst I

CTGCAG

Hinf I

GANTC

11

12/5/2009

Sticky Ends
 Fragments of DNA that has been cut with
restriction enzymes have unpaired
nucleotides at the ends called sticky
ends. All of the fragments will have the
same sticky ends.
 The sticky ends have complimentary
bases, so they could rejoin.

If the vector and the gene to be cloned are both cut

with the same restriction enzyme, they will both have
complimentary sticky ends.

12

12/5/2009

Making Recombinant DNA

To make recombinant DNA, restriction
enzymes are used to cut DNA from two sources
such as the that of a vector and a gene to be
cloned. If the vector and the gene to be cloned
are both cut with the same restriction enzyme,
they will both have complimentary sticky ends
(see above).
After cutting, the two samples of DNA are
mixed. Some of the fragments from one species
will stick to those of the other because they both
have the same sticky ends.

DNA ligase is used to seal the fragments.

Bacteria are capable of taking up DNA
from their environment.
This process is called transformation.
CaCl2 and a procedure called heat shock
are used to make E. coli cells more
permeable so that they take up the
modified plasmids more readily.

13

12/5/2009

Langkah dasar dalam kloning gen

Fragmen DNA yang diklon dimasukkan (insert)
ke dalam DNA sirkular yang disebut vektor
untuk memproduksi chimaera atau molekul
DNA rekombinan
Vektor berfungsi sebagai kendaraan akan
membawa gen ke dalam sel inang, biasanya
berupa bakteri yang mengandung plasmid
atau bakteriophage

Di dalam sel induk, vektor akan mengalami

penggandaan sehingga menghasilkan banyak
duplikat gen dari gen miliknya sendiri dan gen yang
dimasukkan
Pada saat sel mengalami pembelahan maka seluruh
gen yang ada pada sel akan diturunkan pada
keturunannya
Setelah terbentuk koloni besar dari sel-sel inang yang
membawa vektor (pembawa gen asing yang biasa
disebut klon), masing-masing sel akan membawa satu
atau lebih molekul DNA rekombinan. Gen yang
dibawa oleh molekul rekombinan tersebut disebut
klon DNA

14

12/5/2009

DNA rekombinan

Kloning gen

bakteri



Isolasi DNA plasmid dan

DNA yang mengandung
gen yang diinginkan

Sel yang mengandung

gen yang diinginkan

Gen
diselipkan
dalam plasmid


Bakteri rekombinan
Salinan gen diisolasi
dan ditrasfer ke
organisme lain

Plasmid diletakkan
di dalam sel bakteri

Sel diklon dengan

gen yang diinginkan

Identifikasi klon
yang diharapkan

Salinan produk
protein diisolasi

Berbagai aplikasi

Gambaran umum bagaimana bioteknologi menggunakan plasmid bakteri untuk

mengklon gen

15

12/5/2009

Aplikasi Teknologi DNA

Kedokteran dan Farmasi
Diagnosis penyakit
Terapi gen manusia
Produk farmasi

Forensik, Lingkungan dan Pertanian

Forensik
Lingkungan
Pertanian
Peternakan

16