Download Genetic Technology - Solon City Schools

Biotechnology
Biotechnology is the use of
biological processes, organisms,
or systems to manufacture
products intended to improve the
quality of human life.
Genetic Engineering- (A.K.A.
Recombinant DNA Technology)
•  frequency of an allele in a
population
• *involves cutting (cleaving)
DNA from one organism into
small fragments & inserting the
fragments into a host organism
of the same or a different
species
• AMAZING!!! Organism will use the
foreign DNA as if it were its own!!
• Transgenic Organism- organisms that
contain functional recombinant DNA
(rDNA) from a different organism
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4 Areas of Biotechnology
Agriculture
Industry
Forensics
Medicine
Remember DNA?
• What is the monomer of DNA?
– Nucleotides
• How do bases pair?
–A–T
–C–G
• What kind of bond is used?
– Hydrogen bonds between nitrogen bases
I. Restriction Enzymes
• AKA Restriction
Endonucleases
• What macromolecule do
you think they are made
of?
– They are PROTEINS that
cut strands of DNA at
specific nucleotide
sequences
Isolating foreign DNA fragments
• -Restriction Enzymes- DNA cutting
enzymes that can cut both strands of a
DNA molecule at a specific base pair
sequence (A-T, C-G)
• -similar to cutting a zipper into pieces
• -must find the same sequence of base
pairs on both DNA strands but they
must run in opposite directions
Restriction Enzymes (cont.)
A. There are many different restriction
enzymes that each cut DNA at different
nucleotide sequences
B. Most will cut the DNA with a staggered cut
C. Usually occurs at a palindrome: a
sequence of units that can be read the
same way in either direction (ex. Mom,
dad, racecar)
5‘…GAATTC…3’
3‘…CTTAAG…5’
Action of Restriction Enzymes
D. Sticky Ends
1. The staggered cuts leave the DNA with
end pieces “sticking off”
a. We call these “sticky ends”
b. These exposed N-bases will want to join
with other complimentary exposed bases
E. Types of Restriction Enzymes
1. Sticky End- already discussed
2. Blunt End
a. These cut the DNA straight across and
create blunt ends:
CCC GGG
GGG CCC
F. Products generated by restriction
enzymes
1. COHESIVE END CUTTERS (staggered cuts):
Enzyme
Recognition Site
Ends of DNA After Cut
5’…GAATTC…
3’
3’…CTTAAG…
5’
Pst I
5’…CTGCAG…3
’
3’…GACGTC…5
’
2. BLUNT END CUTTERS
(direct cuts):
Enzyme
Recognition Site
EcoRI
HaeIII
5’…GGCC…
3’
3’…CCGG…
5’
5’…G
3’…CTTAA
5’…CTGCA
3’…G
AATTC…3’
G…5’
G…3’
ACGTC…5’
Ends of DNA After Cut
5’…GG
3’…CC
CC…3’
GG…5’
G. Restriction Enzyme Naming
1. Restriction enzymes are named according
to the following nomenclature:
Ex: EcoRI
• E = genus Escherichia
• co = species coli
• R = strain RY13
• I = first enzyme
isolated
How is a transgenic organism
formed??
• Isolate foreign DNA fragment
• Attach DNA fragment to a “vehicle”
(vector)
• Transfer “vehicle” (vector) into a host
organism
Forming transgenic organisms and therefore
clones of genes
Why would anyone go through the
trouble of cutting DNA???
• One reason…
– Recombinant DNA
• Break down the word…what do you think
recombinant means?
• Other reasons…
– DNA fingerprinting, gene therapy…
II. Recombinant DNA
A. Recombinant DNA: DNA that has been
cut from one strand of DNA and then
inserted into the gap of another piece of
DNA that has been broken.
1. The host DNA is often a bacterial cell such
as E coli.
B. Bacterial Structure
1. Bacteria are often used in biotechnology
because they have plasmids
2. A PLASMID is a circular piece of DNA that
exists apart from the chromosome and
replicates independently of it.
3. A plasmid is therefore called a VECTOR.
Vectors transfer DNA
• Vector-means by which DNA from
another species can be carried into
the host cell
• Mechanical Vectors
• Micropipette-inserts into a cell
• Gene guns- tiny metal bullet is coated
with DNA and shot into the cell with a
gene gun
More types of Vectors
• Biological Vectors
• Viruses
• Plasmids-small ring of DNA found in bacteria
cells that is separate from the bacteria’s
normal set of DNA
• Plasmid usually contains genes that may cause
the bacteria to be resistant to certain antibiotics
D. Isolating Genes
1. Must isolate the
gene of interest first
before you insert it
into the plasmid
2. How do you do this?
a. Use a restriction
enzyme!!!
Final Steps of Making Recombinant DNA
1. Once the gene is isolated, have to cut the
organism’s DNA with the same restriction
enzyme…why?
a. The sticky ends will naturally be attracted to each
other
2. Add DNA LIGASE: enzyme that seals the fragments
together
3. After the foreign DNA has been spliced (glued) into
the plasmid using an enzyme DNA ligase, the rDNA
is transferred into a bacterial cell or other organism
4. Now organism is called a Transgenic Organismorganisms that contain functional recombinant DNA
(rDNA) from a different organism
Gene Splicing/Cloning using a
bacterial plasmid
• -IMPORTANT plasmid replicates separately
from the bacterial chromosome & can produce
up to 500 copies per bacterial cell
• -bacteria reproduce quickly (20 min) so a lot of
rDNA is made very fast
• You will essentially be cloning a genegenetically identical copies of rDNA molecules
• -Host cell produces the protein coded for by
the rDNA
III. Uses for Recombinant DNA
A. Recombinant DNA has been gaining importance over
the last few years, and will become more important as
genetic diseases become more prevalent and
agricultural area is reduced. Below are some of the
areas where Recombinant DNA will have an impact:
1.
2.
3.
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8.
Better Crops (drought & heat resistance)
GMO’s (crops like seedless watermelon, pluots, etc.)
Recombinant Vaccines (i.e. Hepatitis B)
Production of clotting factors
Production of insulin
Production of recombinant pharmaceuticals
Plants that produce their own insecticides
Germ line and somatic gene therapy
RECAP
•
Steps for making a
transgenic organism:
1. Locate and isolate the
gene of interest
2. Cut out the gene and cut
the plasmid using the
appropriate restriction
enzyme
3. Insert the desired gene into the plasmid
matching up the sticky ends
4. Use the enzyme DNA ligase to
seal up the sticky ends
5. Transfer the vector in the host organism
where it will replicate
6. Host organism produces the protein coded
for by the recombinant DNA
Insulin Production
Cloning a gene
Transgenic Animals
Cloning an animal
Plants have been genetically
modified to produce insect toxin