Transcript Recombinant DNA and Plasmids

Biotechnology
The use of biological processes,
organisms, or systems to
manufacture products intended to
improve the quality of human life.
Transgenics
• Genetic engineering
makes it possible to
transfer DNA sequences
from one organism to
another
• REMEMBER- DNA is
universal
• A gene taken from one
species can be
replicated in another
species.
Recombinant DNA
• Recombinant DNA contains DNA from
2 or more different sources.
• So how does DNA get from one species
to another?
• Vector: a mechanism needed to transfer
DNA from one organism to another
– Microinjection
– Gene Gun
– Bacteria Plasmid
– Virus
Recombinant DNA
• The host cell is often a bacteria cell
• Bacteria are used because they have
plasmids
• A plasmid is a circular
piece of DNA that exists
apart from the
chromosome and
replicates independently of it.
• So how do I take a gene from one
organism and put it into another?
• We need to use Restriction
Enzymes
What are Restriction Enzymes?
• Proteins
• Used by bacteria to cut viral DNA
• They “restrict” the growth of viruses
• What does this have to do with
transgenics?!
Restriction Enzymes
• Cut strands of DNA at specific nucleotide
sequences
• There are many different restriction
enzymes that each cut DNA at different
nucleotide sequences
• Usually occurs at a palindrome
5’ GAATTC
3’ CTTAAG
Sticky Ends
• Most restriction enzymes cut
DNA with a staggered cut
• The staggered cuts leave
the DNA with end pieces
“sticking off”
– We call these “sticky ends”
– These exposed N-bases will
want to join with other
complimentary exposed
bases
So what ?
• What do you predict could happen if two
pieces of DNA are cut with the same
restriction enzyme???
– YES! They will have the same “sticky ends”
– How could we use this???
• Restriction
Enzymes can be
used to make
Recombinant
DNA.
• The gene of
interest can be
isolated using
Restriction
Enzymes
Making Recombinant DNA…
• Once the gene is isolated, how do we join
it with the organism’s DNA?
• Cut the organism’s DNA with the same
restriction enzyme…why
– The sticky ends will naturally be attracted to
each other
• Add DNA LIGASE: an enzyme that seals
the fragments together
• Now whenever the bacteria plasmid
replicates, the gene will be cloned.
• Gene Cloning: the production of many
identical copies of the same gene.
• You have created a
Transgenic Organism!
–organisms that contain functional
recombinant DNA (rDNA) from a
different organism
• http://www.sumanasinc.com/webcontent/a
nimations/content/plasmidcloning.html