Tugas Kapsel

Tugas Kapsel
Cesis Rahmi Riana Sani

260110110085
History of Recombinant
DNA Technology
History of Recombinant DNA Technology

In 1970s, Paul Berg created a piece of recombinant DNA
by joining together DNA from E. coli chromosome and
DNA from simian virus 40 (SV40), a primate virus.
Berg isolated both DNA samples, cut with EcoRI, added
to a reaction tube with enzyme DNA ligase a hybrid
molecule
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In the same time, Stanley Cohen and Herbert
Boyer worked with two bacterial plasmid.
Cohen postulated that plasmids could be

used as vectors : pieces of DNA that can
accept, carry and replicate other pieces of
DNA
They cut both plasmid with EcoRI, then
joined fragments together using DNA ligase
to created a recombinant plasmids
Product : pSC101 (cloning vector) contained
a gene for tetracycline resistance and
restriction sites for several enzymes including
EcoRI and HindIII

DNA from Xenopus laevis clone into pSC101 ligation of
DNA fragments transform into bacteria with calcium
chloride solution

In 1974, gene cloning pioners and cloning critics voiced
concerns about the safety of genetically modified
organism (GMO)
In 1975, an invited group of wellknown molecular
biologists, virologists, microbiologists, lawyers and
journalists gathered at the Asilomar Conference Center
in Pasific Grove, California
Result : National Institutes of Health (NIH) form the
recombinant DNA Advisory Committee (RAC)
In 1976, the RAC published a set of guidelines for
working with recombinant organisms
Recombinant DNA Technology

Preparation of pure samples of
DNA :
- total cell DNA
- vector DNA : plasmid, etc.
Cutting DNA molecules with
restriction enzymes
Analysis of DNA fragment sizes
(by electrophoresis)
Joining DNA molecules together
with DNA ligase
Introduction of recombinant DNA
(chimaera) into host cells
Selection of cells that contain
recombinant DNA
Preparation of pure samples of DNA:
Total cell DNA
Total cell DNA may be DNA from

a culture of bacteria, a plant
cells, an animal cells, etc.
Preparation of Total Cell DNA

1. Growing and harvesting a cell culture
2. Disruption of cell wall and membranes to liberate
cellular components.
3. Inactivation of DNA and RNA-degrading
enzymes (DNases and RNases).
4. Separation of nucleic acids from other cellular
components.
5. Concentration of the resulting DNA solution
Preparation of pure samples of DNA:
Vector DNA
Vectors DNA
Vectors DNA :
- a vehicle that transport the gene into a host cell or
- a piece of DNA that can accept, carry and replicate
(clone) others piece of DNA
Many types of DNA vectors :

Plasmids
Bacteriophage vectors
Cosmid vectors
Bacterial artificial chromosomes (BACs)
Yeast artificial chromosomes (YACs)
Human artificial chromosomes (HACs)
Ti vectors
Plasmid Isolation
The first step in making recombinant DNA is to isolate
donor and vector DNA. General protocols for DNA isolation
were available many decades before the advent of
recombinant DNA technology.
With the use of such methods, the bulk of DNA extracted
from the donor will be nuclear genomic DNA in eukaryotes
or the main genomic DNA in prokaryotes; these types are
generally the ones required for analysis.
The procedure used for obtaining vector DNA depends on
the nature of the vector. Bacterial plasmids are commonly
used vectors, and these plasmids must be purified away
from the bacterial genomic DNA. A protocol for extracting
plasmid DNA by ultracentrifugation
Cutting DNA
molecules
Cutting DNA molecules
Analysis of DNA
fragment sizes
Gel Electrophoresis
Gel Electrophoresis
17
Measuring fragment size

compare bands to a known standard
usually lambda phage virus cut with HindIII
nice range of sizes with a distinct pattern
Joining DNA
molecules together
Paste DNA
DNA Ligase
enzyme seals
strands
bonds sugarphosphate bonds
covalent bond of
DNA backbone
can joined together
DNA with cohesive
ends or blunt ends
Introduction of
recombinant DNA into
host cells
Transformation
Transformation : a process for inserting foreign DNA
into host cell
Types of transformation mechanism :
1.Natural transformation : Bacillus
subtilis,Streptococcus pneumoniae, Haemophilus
influenzae, Neisseria gonorrhoeae
2.Artificial transformation
Artificial Transformation
Transformation method :
1. Calcium chloride treatment
2. Electroporation
3. Microinjection/Gene gun
4. Agrobacterium-mediated transformation

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Uploaded by

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Tugas Kapsel

Cesis Rahmi Riana Sani

History of Recombinant DNA Technology

History of Recombinant DNA Technology

Cohen postulated that plasmids could be

History of Recombinant DNA Technology

History of Recombinant DNA Technology

Recombinant DNA Technology

Preparation of pure samples of DNA:

Total cell DNA

Total cell DNA may be DNA from

Preparation of Total Cell DNA

Preparation of pure samples of DNA:

Many types of DNA vectors :

Cutting DNA molecules

Measuring fragment size

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