Download Recombinant DNA and Cloning

Recombinant DNA and
Cloning
Riyanda N G (10198)
Vina E A (10221)
Arini N (10268)
Suluh N (10302)
Cloning Definition
The word "clone" has several different
meanings in biology. As a noun, a clone is
an identical genetic copy of either a piece
of deoxyribonucleic acid (DNA), a cell, or a
whole organism. Identical twins are
clones, as are two daughter cells
produced by mitosis. As a verb, "to clone"
means to produce identical genetic copies
of either pieces of DNA, cells, or whole
organisms.
DNA cloning
DNA cloning is usually performed for one of
two reasons: either to produce a lot of
identical DNA for further study, or to use
the DNA in an intact organism to produce
useful proteins.
• Recombinant DNA – technique allowing joining two different
DNA molecules, amplify them and modify them
• Allow isolation of piece of DNA out of genome and
amplification of DNA fragment
• Restriction endonucleases and DNA ligases
EcoRI
EcoRI
Mixing together and annealing
Recombinant DNA
Techniques For Cloning
• Isolate and purify all the DNA from a sample of human
cells. Break apart the cells and then wash, centrifuge,
and use other purification techniques.
• • Cut the DNA into millions of small fragments using
restriction enzymes. Each DNA piece may be as large
as 10 kb, but is more commonly 1 to 5 kb.
• • Mix the DNA fragments with plasmids that have been
cut with the same restriction enzymes. Add DNA ligase,
an enzyme that joins the human DNA fragments to the
plasmids and seals the circles up again. By using the
right ratio of plasmid to fragment, a researcher can
ensure that each plasmid harbors at most one human
DNA fragment. With luck, one DNA fragment will contain
the insulin gene.
• • Cause a bacterial culture to take up the
plasmids. This can be done by ionic shock.
Again, adjusting the ratio can ensure one
plasmid per bacterium. The plasmid used
usually carries a gene for antibiotic resistance.
• • Grow the bacteria on antibiotic-containing agar
plates, spread very thinly. The antibiotic will kill
bacteria that didn't take up the plasmid. Single
bacteria give rise to colonies, which will appear
as small spots on the plate. The resulting
bacterial colonies are called a genomic library.
• • To find which of the colonies includes the human insulin
gene, use a probe. This is typically a radioactive
segment of DNA whose sequence is complementary to
part of the insulin gene, allowing it to bind. Apply the
probe, and see where it sticks.
• • Isolate that colony, and let it multiply in a rich broth.
Each bacterium will replicate the insulin gene, providing
many copies to work with. Including the appropriate
promoters and other regulatory factors will prompt the
bacteria to synthesize the human insulin protein, which
can then be purified for medical use.
Construction of a recombinant DNA molecule
• Recombinant DNA vectors:
– Amplification of DNA fragment can be achieved in the cell
using cloning vectors: plasmid or bacteriophages
– Plasmid Small circular DNA in bacteria or yeast cells
Accumulate 1-5 kb inserts
LacZ encodes -galactosidase
Lacl – encodes factor controling
transcription of lacZ
Bacterial transformation
Introduction of DNA into bacteria
Spontaneous uptake – low probability
E. coli – cells treated with CaCl2
Less than 1 of 103 cells acquire a
plasmid
Selection of transformed cells:
resistance to antibiotics
using chromogenic substances
Antibiotics: molecules produced by
microorganism that kill other
microorganism
peniciline, tetracycline, ciplroflaxine –
inhibits gyrase in the complex with DNA
– inhibits DNA replication
Chromogenic substances:
Bacteriophage
Bacteriophages are bacteria viruses.
When they infect their host they use host
machinery to replicate their DNA.
In bacteriophage vectors part of the phage
DNA is replaced by the gene of interest
(max. size is 15-25 kbp)
When this new recombinant phage DNA
infects a host the gene of interest will be
replicated.
commonly used phage vectors include
M13 and λ.
Cloning DNA in bacteriophage λ
Infection of bacterial cell:
Tail sticks to the cell wall
DNA from head is squired
into bacteria
Phage genes are
transcribed by bacterial
RNA polymerase
Corresponding mRNA is
translated using bacterial
machinery
Newly replicated phage
DNA and head and tail
protein ensemble
spontaneously
Lyses of host bacteria
results in formation of
plagues.
• Screening
– In situ hybridization
Protein expression
- Gene is inserted into
plasmid
- Plasmid is transformed
into
a host cell (E. coli)
-Cell culture is prepared
-Each cell contains several
copies of the plasmid with
gene
-Gene expression leads to
the production of protein
-Protein level may reach
-30% of total cellular
protein
-Isolation of protein
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