Download Gene Cloning

Recombinant DNA Technology
“Gene Cloning”
What is it?
 Gene cloning: production of large quantities of a specific,
desired gene or section of DNA to produce identical copies
 Cloning requires the use of a vector which is a self replicating
molecule that can replicate inside the host organism.
 The two main kind of vectors are Plasmids and
bacteriophages
- The Purpose of gene cloning is to yield large quantities of a
particular gene or its protein product.
1. Plasmid obtained from bacteria
3. Isolated DNA is
inserted into plasmid
with DNA ligase
Antibiotic resistance gene is also added
to plasmid to act as a marker
A marker gene is also added to the
recombinant DNA Lac-c
2.Gene containing foreign DNA is isolated
using restriction enzymes
4.Plasmids are then inserted
back into the bacteria
(transformation)
5. Bacteria then multiple, producing identical co
of the “foreign DNA” i.e. Clones
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Two marker genes an antibiotic resistance gene and a second marker
gene Lac-c
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Recombinant DNA planted on agar containg x-gal and an antibiotic
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Lac-c codes for beta galactosidase enzyme which catalyzes a colourless
chemical x-gal to form a blue compound. Thus would appear blue on
agar.

If the Lac-c gene is disrupted by the insertion of the isolated gene beta
galactosidase enzyme can not be produced. Thus colonies will stay white
on agar

bacterial cells on agar containing antibiotic and X-gal any colonies that
grow and are white contain our recombinant DNA with our gene of
interest.
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The gene you want to be cloned
Bacteria can’t remove introns so we need to
carry out Reverse transcription
mRNA from gene of interest and make DNA
using reverse transcriptase, an enzyme which
allows a complementary strand to be formed
using mRNA as a template.
1. Plasmid obtained from bacteria
3. Isolated DNA is
inserted into plasmid with
DNA ligase
2.Gene containing foreign DNA is isolated
using restriction enzymes
4.Plasmids are then inserted back
into the bacteria (transformation)
5. Bacteria then multiple, producing identical copies
of the “foreign DNA” i.e. Clones
Bacteria then plated onto an
agar plate containg X-gal and
an antibiotic.
Only bacteria that grow are the
ones that received wanted DNA
Prepare gene of interest mRNA to DNA
Restriction enzymes to cut DNA plus plasmid
Marker genes, antibiotic resistance and lac-c are
added
 DNA ligase to attach isolated DNA with plasmid
 Insertion back into host bacterium
(transformation)
recombinant DNA
 Replication
 Screening is then carried out to find which cells
contain the plasmids with the gene of interest.
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Host Bacterium
Phage attaches to Bacteriu
and injects its DNA
Bacteriophage
Phage uses bacterial metabolism
to make more phage particles
•
Reproductive cloning for endangered species
•
Therapeutic cloning technology may some
day be used in humans to produce whole
organs from single cells or to produce healthy
cells to replace degenerative cells
•
Resistance to disease or improve nutritional
quality