Download Recombinant DNA

Chapter 14
DNA Technologies
Recombinant DNA
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Recent technology – still changing
New combinations of DNA – usually a DNA
sequence inserted into bacteria
Allows DNA sequence to be copied and
amplified (a.k.a. cloned)
Cloned DNA
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Clone – a collection of molecules or cells, all
identical to an original molecule or cell
To clone a gene – make many identical copies of it,
often by placing it in a culture of bacteria
The cloned gene can be a normal copy (‘wild type’)
or an altered version (‘mutant’)
Recombinant DNA technology makes gene cloning
possible
The goals of gene cloning are: study of specific
genes and genetic engineering
Overview: Gene Cloning
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Restriction enzymes are used to cut DNA
molecules in specific places
The fragments of DNA are incorporated into
a vector, such as a bacteriophage or plasmid,
which can carry it into a host cell
The recombinant DNA is replicated and
distributed to daughter cells during cell
division
Step 1: Restriction enzymes
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Enzymes found in bacteria – are helpful to them to fight
foreign, invading DNA
There are thousands of different restriction enzymes
These enzymes cut DNA at specific sites
These cleavage sites are usually at a 4 or 6 base-pair
palindromic sequence
The ‘top’ strand from 5’ to 3’ is the same as the ‘bottom’
strand from 5’ to 3’
Cleavage that cuts the two strands directly across from each
other leaves blunt ends
Cleavage that is uneven leaves single-stranded tails called
sticky ends
Restriction enzymes…
Cuts usually occurs at
a palindromic sequence
SmaI: produces blunt ends
5´ CCCGGG 3´
3´ GGGCCC 5´
EcoRI: produces sticky ends
5´ GAATTC 3´
3´ CTTAAG 5´
Examples of Palindromes:
Don't nod
Dogma: I am God
Never odd or even
Too bad – I hid a boot
Rats live on no evil star
No trace; not one carton
Was it Eliot's toilet I saw?
Murder for a jar of red rum
Some men interpret nine memos
Campus Motto: Bottoms up, Mac
Go deliver a dare, vile dog!
Madam, in Eden I'm Adam
Oozy rat in a sanitary zoo
Ah, Satan sees Natasha
Lisa Bonet ate no basil
Do geese see God?
God saw I was dog
Dennis sinned
Restriction enzymes…
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The names are derived from the names of the bacteria they
originally came from
They always cut DNA in the same place, regardless of the
source of the DNA
Enzymes with staggered cuts  complementary ends
(overhangs are ‘sticky’)
These result in complementary ends that can be ligated
together
Enzymes that cut at the same position on both strands leave
blunt ends
DNA fragments with blunt ends can also be ligated together,
but with lower efficiency
Step 2: Incorporation into vectors
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Plasmids are cleaved by restriction enzymes
and have sticky or blunt ends
Foreign DNA fragments (cut in the same
places so they have the same ends) can be
linked to the ends with DNA ligase and
inserted into the plasmids
This produces the recombinant DNA
Useful vectors…plasmids
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Plasmids are naturally occurring extra-chromosomal
DNA molecules found in bacteria
They are circular and double-stranded
They are the means by which antibiotic resistance is
often transferred from one bacteria to another
(remember the mice in Griffith’s experiments?)
They do not usually contain genes essential to the
bacteria under normal conditions
Useful vectors… viruses
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Bacteriophages are viruses that infect
bacteria
These can also be made to carry recombinant
DNA into bacteria for cloning
Engineered viruses can also be used as
vectors into cells of eukaryotic organisms
Step 3: Replication of Recombinant
DNA
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The cells containing the recombinant DNA are
grown in culture – replicating the new DNA as they
do
Not all the cells will be descendants of those with the
recombinant DNA  these need to be eliminated
from the culture
For this reason, plasmids that also confer resistance
to a particular antibiotic or allow cells to use a
specific nutrient are often used
This allows scientists to separate transformed cells
from untransformed cells
Why recombinant DNA?
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This technology has been used to:
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Add favorable genes to agricultural crops, for
example pest or herbicide resistance
Modify bacteria to produce enzymes used in
industry, for example the enzyme used for
making cheese
Produce therapeutic products such as human
insulin and blood clotting factors
In the future we hope to:
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Provide gene therapy – the goal is to introduce the
correct version of a gene into the cells of a patient to
correct protein production
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sickle-cell, cystic fibrosis, and some eye diseases
Silence overactive genes through RNA interference
(RNAi) – uses a double-stranded RNA molecule to
stop protein production of a targeted gene
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lupus, Crohn’s disease, autism, Alzheimer’s, rheumatoid
arthritis, Parkinson’s disease
Other DNA Technologies
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Polymerase Chain Reaction (PCR)
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Uses nucleotides and primers, along with heat, to copy
tiny amounts of DNA many times in vitro
Useful for analysis of crime scene evidence or other times
when additional DNA is desired
Sensitive to contamination from outside DNA
Gel electrophoresis
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Allows us to separate DNA fragments based on their
movement through a gel submitted to an electrical field
The smallest fragments of DNA migrate faster (and
therefore further) from the starting point
This is the basis for ‘DNA fingerprinting’