Download Recombinant Biotechnology

Genetic Research and Biotechnology
Recombinant technology
Recombinant DNA technology
Selection of Traits
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Artificial selection - breeding; novel crosses
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Recombinant DNA technology - DNA from
one organism cut and spliced (recombined)
with DNA of another
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Genetic engineering - gene isolation,
modification and insertion into original or
different organism
Recombinant DNA technology
Recombinant DNA - DNA which has been altered by joining genetic
material from two different sources. It usually involves putting a gene from
one organism into the genome of a different organism, generally of a
different species.
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‘Typical ‘ bacteriophages have a hexagonal
head, tail and tail fibers
Attach to cell wall of a host cell with tail fibers
Alter the cell wall of host cell inject nuclear
material into host cell
Werner Arber proposed that restricted growth of phages occurred because some
bacteria contained enzymes that could cut viral DNA into small pieces, thus
preventing viral replication.
http://www.pbs.org/faithandreason/biogloss/recomb-body.html
Bacterial and Viral Battles
• Some bacteria and viruses insert their DNA
into other cells that are to act as hosts
• Bacteria use restriction enzymes to cut up
foreign DNA
• These characteristics are useful in recombinant
technology
Roots of Recombinant Technology
• Plasmids are small, circular
pieces of DNA in bacteria,
that replicate
independently of the
bacterial chromosome
• Pieces of foreign DNA can
be added to plasmids to
create recombinant DNA
• Replication can produce 50
– 100 copies of the
recombinant plasmid
Recombinant DNA technology
Restriction enzymes cut DNA by cleaving the phosphodiester bond in the DNA strand.
• Like other enzymes restriction enzymes show specificity for certain substrates, and will
only digest DNA within specific sequences of bases - called recognition sequence or a
restriction site.
• Some restriction enzymes cut DNA into overhanging single stranded ends.
• Others will generate fragments with double-stranded non-overhanging ends called
‘blunt ends’.
Recombinant DNA technology
Recombinant DNA refers to DNA which has been altered by joining genetic
material from two different sources. It usually involves putting a gene from
one organism into the genome of a different organism, generally of a
different species.
DNA from any source such as bacteria, humans, dogs, cats, frogs, dinosaurs, can
be digested.
How would you put cut DNA back together??
DNA ligase
Paul Berg founded recombinant DNA technology when he spliced together DNA
from E.coli chromosome and DNA from a primate virus.
Recombinant DNA technology
Restriction enzymes
cut double stranded
DNA at specific sites
These cuts produce
DNA fragments that
can be fit back
together
Ligase is like glue, and
rejoins the DNA.
Cohen also made another
important contribution to
gene cloning – he
demonstrated
transformation:
a process for inserting
foreign DNA into bacteria.
•use of calcium chloride
solution and temperature
change, or
electroporation
•compromise the
bacterial membrane
enough to allow plasmid
DNA to enter
the cells.
Antibiotic selection can be used to select the transformed gene.
Summary
We can :
• cut DNA (what do we use??)
• paste it back together (what do we use??)
• put it into a vector (what does this mean??)
• get the vector into a cell of interest(why)??
So what?? How would you use this??
Gene Cloning in Bacteria
Cloning
…specialized DNA technology to produce multiple,
exact copies of a single gene or other segment of DNA
to obtain enough material for further study.
Requires a Vector…
...a specialized DNA sequence that can enter a living cell,
...signal its presence to an investigator,
... and provide a means of replication for itself and the
foreign DNA it carries.
And, Vectors…
…contain unique restriction
sites to facilitate the
creation of recombinant
DNA molecules,
... must also possess a
distinguishing physical
characteristic such as size
or shape by which it can
be purified away from the
host cells genome.
Step 1…restriction digests and ligation of fragments
into cloning vectors,
Cloning Step 2
…vector-insert recombinants are inserted in
host cells,
– Transformation (via bacterial mechanisms)
– Transduction (via viral mechanisms)
Cloning Step 3
...vectors contain selectable
markers,
– only cells that contain
vector DNA will survive
selection,
– recombinant vectors can
be discerned from empty
vectors by additional
markers.
Blue/White Cloning
Amplification
…if you were to grind me up with a giant
mortar and pestle, and extract all of my
DNA coding for a single gene, you’d get
about 1 mg of DNA,
…two liters of bacterial culture carrying my
gene in a recombinant DNA vector, would
yield an equivalent mass of DNA.
Bacterial Production of Human Insulin
• The final steps are to collect the bacteria, break open the
cells, and purify the insulin protein expressed from the
recombinant human insulin gene