Download Biotechnological Tools and Techniques

Biotechnological Tools
What are we doing here?!?!
One of the major advances in genetic research is the
usage of recombinant DNA.
Recombinant DNA refers to the practice of placing DNA
(genes) from one organism into the genome of a second
organism in hopes that the second organism will make
use of the genes and make the proteins for which the
genes encode.
Basically, you take a desired gene from one thing and
stick it into another so it too will be able to make the
desired protein from the inserted gene.
To understand and perform any biotech work you have to
know the tools and the procedures needed in order to get
the job done. So let’s take a look…
Tools of the Trade
Restriction Endonucleases
AKA – Restriction Enzymes
These enzymes cut DNA at specific sequences called
recognition sites.
The cuts at the recognition sites can be in the form of
either blunt ends or sticky ends (with sticky ends being
the more useful of the two).
Restriction enzymes naturally occur in bacteria as a
defense against viral infection. The virus would inject its
DNA into the bacterial cell only to be cut into many
pieces and be rendered useless.
Sticky vs. Blunt Ends
There are two possibilities when a restriction
enzyme cuts through the DNA.
1.
2.
Sticky Ends – Staggered ends on a DNA molecule with
short, single-stranded overhangs.
Blunt Ends – A straight cut, down through the DNA that
results in a flat pair of bases on the ends of the DNA.
Sticky ends are preferred in the lab because the
single-stranded overhangs are complementary to
each other and can be stuck back together with
other sticky ends made from the same restriction
enzyme.
Blunts ends can match up with any other blunt end
so you don’t get that exact match between the two
pieces of DNA you are trying to put together.
Sticky Ends vs. Blunt Ends
Methylases & DNA Ligase
Methylases are enzymes that add a methyl group
(–CH3) to a nucleotide in the recognition site of the DNA.
This extra methyl on the nucleotide changes the shape
of the recognition site and the restriction enzyme is
unable to cut it because of the change in shape.
Methylases are also naturally found within bacteria – it is
how they protect their own DNA from their restriction
enzymes.
DNA Ligase reforms the phosphodiester bonds between
adjacent nucleotides when you are trying to connect the
foreign DNA fragment and the host cells DNA.
Gel Electrophoresis
Gel electrophoresis is a technology that separates
molecules based on charge and size by sorting them
in a gel meshwork.
The gel (meshwork) is like the rubber band tunnel at
McDonald’s. You know the one – that thin, long, trap
with those thick black bands running through it this
way and that. You have to really squirm your way
through it to get to the other side.
Well…The smaller you are the easier it is to fly
through it. And vice-versa of course.
The charge on the molecule dictates the direction the
molecule moves within the gel. Opposite charges
attract so positive molecules move to the negative
electrode while negative molecules move to the
positive electrode.
Tasing the DNA
DNA has a negative charge – all those phosphates in the
backbone!
We use that property to make the DNA move. An electric
current is passed through the gel and charged particles
(like DNA) will move in response to that electric current.
Your DNA samples are loaded into wells (small pits)
within the gel. The wells for DNA are near the negative
electrode so they will move to the positive electrode –
opposites attract!
When the switch is thrown - the DNA fragments move.
The smaller the fragment, the further it moves within the
gel.
After the gel has been run, it is treated with ethidium
bromide that clings to rungs of the DNA and causes it to
fluoresce (glow) under a UV light.
Gel Electrophoresis
Plasmids
Plasmids are small, circular pieces of DNA that can exit
and enter bacterial cells.
They contain “bonus” DNA in that they can have genes in
them that allow the bacterial cell to become resistant to
some of the things that would normally kill it. These genes
are known as resistance genes.
We can insert foreign DNA into plasmids and put them
into bacterial cells for them to use.
We use the resistance genes to show us whether or not a
plasmid has successfully adopted a plasmid that contains
the foreign DNA. The foreign DNA interrupts one of the
resistance genes on the plasmid and the bacteria that has
that plasmid loses its ability to be resistant to whatever
substance it provide the resistance against.
Plasmid Pics
Transformation
The introduction of foreign DNA into a bacterial
cell is known as transformation.
Transformation requires a vector (or delivery
vehicle) that will bring the foreign inside the
bacterial cells. Plasmids or viruses are normal
vectors for transformation.
Any cell that has been successfully transformed
and that now contains the foreign DNA is known
as a competent cell.
To Sum It All Up…
These are the basic
tools needed to
perform any
experiments or work
in a biotech lab.
Future lectures will
deal with some of the
techniques and
processes that are
employed in these
same labs.
FIN