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BIOTECHNOLOGY
Recombinant DNA
• Recombinant DNA: a fragment of DNA composed of
sequences originating from at least 2 different sources
• Why? – ex: Agricultural Benefits: increase in crop yields;
disease resistance; crop longevity
Genetic Engineering
• HOW??
• Hypothetically…..
• If we could insert a gene into another organism’s genome
(DNA), that organism would express that gene (make the
protein the gene codes for)
• To do this, we would need “molecular scissors” to cut the
gene sequence from our original source and “molecular
glue” to insert the gene sequence into the host organism’s
DNA
Restriction Endonucleases
• Also known as Restriction Enzymes are “molecular
scissors” that can cleave (cut) double stranded DNA at a
specific base-pair sequences.
• Bacterial enzymes
• Recognition Site: the specific sequence where the
restriction enzyme makes its cut.
• Usually palindromic
• ~4 to 8 nucleotides long
STICKY ENDS
• STICKY ENDS: both fragments of the newly cleaved DNA
have DNA nucleotides lacking complimentary bases.
• Sticky end fragments make use of hydrogen bonding, thus
facilitating the initial joining of fragments that then are completely
joined by another enzyme
• BLUNT ENDS: The ends of the DNA fragments are fully
paired.
• Blunt ends have a significantly lower yield and have the potential to
insert DNA in the opposite orientation desired
• Restriction enzymes that produce sticky ends are more
useful because these DNA fragments can easily be joined
to other DNA sticky ends fragments made by the same
restriction enzyme.
• Can easily be used to create recombinant DNA
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::5
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bio37.swf::Restriction%20Endonucleases
Plasmids for Genetic Engineering
A desired sequence of nucleotides from source DNA can be cut out of
the genome, separated and annealed to another DNA molecule
These DNA fragments can be inserted into bacterial plasmids
Plasmids are small, circular, double stranded DNA molecules found in
bacterial cells
These plasmids are independent of the chromosome of the bacteria
The DNA contained in plasmids can be replicated and expressed
When foreign DNA is taken up by a bacterial cell via plasmid or virus
this is known as TRANSFORMATION.
Restriction sites
The DNA that
we want to
insert the gene
into
The gene
we want to
introduce
The gene is
cut from its
original DNA
strand
Application – The Ti Plasmid
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::5
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bio40.swf::The%20Ti%20Plasmid
DNA Ligase
• The same restriction endonuclease (enzyme) must be
used on both DNA otherwise the 2 DNAs won’t be able to
bind together – there needs to be complimentary base
pairing (the sticky ends must match up)
• Two complimentary strands will naturally anneal and form
hydrogen bonds between the base pairs but an enzyme is
required to form the phosphodiester backbone of the DNA
• DNA Ligase is used to join the cut fragments of DNA
together via a condensation reaction. Phosphodiester
bonds are reformed between adjacent bases
• T4 DNA Ligase – is specific enzyme that is especially
useful for joining blunt ends together
Technique for Gene Transfer
• Materials Required:
• A vector (used to carry the gene into the host
cell)
• A host cell (which will express the gene – make
the protein)
• Restriction enzymes
• DNA ligase –(used to join together DNA blunt or
sticky ends)
Animation
• Frog experiment
• http://highered.mcgraw-
hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/
dl/free/0072437316/120078/bio38.swf::Early%20Genetic
%20Engineering%20Experiment
WHERE DO RESTRICTION
ENZYMES COME FROM?
Restriction enzymes act as an immune system in the
bacterium. When a bacteriophage (a virus) tries to
inject its DNA into the bacteria, restriction enzymes
cut up the bacteriophage DNA into many fragments
– thus, preventing it from doing any harm to the
bacterium.
Methylases
• Restriction endonucleases must be able to distinguish
between foreign DNA and their own DNA otherwise they
would cut up their own DNA.
• METHYLASES are enzymes that modify a restriction site
by adding a methyl group to and preventing the restriction
endonuclease from cutting it. This prevents the cell from
cutting its own DNA.
methylation
• Foreign DNA in a cell will not be methylated and therefore
may be broken down by the enzymes
Real Life Application: INSULIN
• Insulin is a hormone that regulates blood sugar levels by
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converting excess glucose into glycogen for long term
storage.
Diabetics do not make sufficient amounts of insulin.
Diabetics may be required to take insulin injections.
Using restriction enzymes, the gene for synthesizing
insulin is cleaved out of DNA and inserted into the DNA
plasmid of a nonharmful bacteria.
DNA ligase is added to the bacteria.
The bacteria is also given the necessary amino acids.
The recombinant DNA will express the insulin gene and
make insulin that can be collected and administer to
diabetics
Human Genome Project
• Scientists worked to sequence a chromosome, one gene
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at a time and pooled the information together and store
this information in database.
The aim was to;
determine the sequences of the 3 billion chemical base
pairs that make up human DNA.
identify all the approximate 30,000 genes in human DNA.
Have a map of the sequence of nucleotides of human
DNA that could lead to mapping of genes (listing and
finding the locus of each human gene)
Human Genome Project: OUTCOME
• Improved understanding of genetic diseases
• Production of medicines (based on DNA sequences) to
cure diseases
• Genetic screening (and preventative medicine)
• Focused research
• Provides more information about the evolutionary paths
between species
Human Genome Project
• NOVA Online | Cracking the Code of Life | Watch the
Program Here
• genome.gov | Education Kit - Component DOWNLOAD
Page
• Sequence for yourself
• http://www.pbs.org/wgbh/nova/body/sequence-DNA-for-
yourself.html