Biotechnological Tools and Techniques

Biotechnological Tools and Techniques
Molecular Geneticists use many different types of tools in order to aid in the
investigation of genetic disorders, alteration of genetic makeup of many
organisms, or anaylze the samples collected in criminal investigations.
Restriction Endonucleases (restriction enzymes)
These are enzymes that cleave (or cut) the double stranded DNA molecule into fragments at specific
points.
There are many different types of restriction enzymes
formed by various Bacteria that can be used to cut the DNA
strands at different locations. Bacteria use this ability to
protect themselves from invading viruses by chopping
them into pieces when they enter. These diagrams show
EcoRI restriction e
Each type of restriction enzyme will recognize a particular set of
nucleotides. This is known as the recognition site.
Restriction enzymes are named by the following things:
HindIII
H represents genus Haemophilus
in-­‐represents species influenza
d represents the strain Rd
III the third restriction enzyme isolated from this strain

Methylases
These are specific enzymes that add a methyl (CH 3 ) group to the recognition
site to prevent a restriction enzyme from attaching and cleaving a DNA
fragment into pieces.
DNA Ligase
This enzyme is used by the DNA to join fragments together. It is used during DNA replication as well as
after restriction enzymes cut apart different pieces of DNA.
Gel Electrophoresis
Once pieces of DNA have been cut out of the full
DNA molecule, they can be separated and sorted on
the basis of size.
DNA is negatively charged because of the phosphate
group.
The larger the piece of DNA the greater the size and
negativity of the molecule.
Electrodes are hooked up to the sample and the
negatively charged particles move toward the
positive + electrode. The smaller the piece, the
farther it will move.
Each sample that is gathered will be analyzed using restriction enzymes (cut into
pieces) and then run through gel electrophoresis.
Samples that match or people who are related will have matching fragments of
DNA.
Who is guilty of this crime?

Plasmids
Plasmids are small circular piece of DNA that can exit and enter
bacterial cells.
Bacteria cells can incorporate Plasmids, and express the proteins
that are coded for in the plasmids.
Insulin creation
Originally diabetic patients required either insulin harvested
from cadaver pancreases or insulin from bovine or pig source.
Once Plasmids were discovered bacterial plasmids were
inserted with the human insulin gene. The bacteria created the
insulin through Protein synthesis. The human insulin is then
harvested by chemical processes.
Transformation
The introduction of foreign DNA usually as a plasmid or virus, into a bacterial cell. Plasmids can be used
as vectors (a method of transferring DNA into a host cell). If a bacterium will accept a plasmid it is called
a Competent Cell. Most cells can be made to be competent by chemically inducing with Calcium
Chloride.