Biotechnology:

1
Biotechnology

• Or how I stopped worrying and learned to love
  the sheep.

2
Restriction Enzymes

• Restriction enzymes are compounds first isolated
  in the 1970's
• They function by selectively cutting DNA at
  specific sequences

3
Restriction Enzymes

• These cuts usually occur in the following forms.
• The cut can be made straight across a base-pair
  sequence resulting in a "Blunt End
• The cut can be made in an offset manner leaving
  exposed nucleotide sequences. These exposed
  sequences are called "Sticky Ends"

Blunt End
Sticky end
4
Gene Splicing

• The presence of sticky ends allows segments of
  DNA to be joined together. Since DNA strands
  which have been cut by the same restriction
  enzyme can easily bond together according to base
  pairing rules.

5
Gene Splicing contd..

• This allows for genes to be "cut pasted"
  between organisms. This can be seen with
  production of human insulin.
• The DNA sequence of insulin is identified and cut
  out using a restriction enzyme.
• A plasmid from E. coli is removed and cut open
  using the same restriction enzyme
• Since both fragments have complimentary sticky
  ends the bind and the gene for human insulin is
  integrated into the plasmid
• The plasmid is then reinserted into a bacterial
  cell. This cell will produce insulin and is
  cultured. Human insulin can now be extracted and
  provided to diabetics.

6
(No Transcript)
7
Gel Electrophoresis

• Gel electrophoresis is a technique used to
  separate fragments of DNA.
• Separates fragments as a function of size.
• Most types use Agarose to separate fragments.
• Agarose is a porous gel. It can allow the
  passage of molecules through, however, larger
  molecules move more slowly through it since they
  cannot squeeze through the pores as easily as
  smaller molecules.

Electrophoresis Apparatus
8
Electrophoresis Technique

• An agarose gel is casted with several holes
  called wells at one end.
• The gel is placed in an electrophoresis box which
  is filled with an electrolyte buffer solution.
• Samples of digested DNA are placed in the wells
• Electrical leads are attached to the ends of the
  box creating an electrical potential across the
  apparatus.
• Because DNA has a negative electrical charge. It
  is "pulled" towards the positive side of the
  apparatus.
• Also, since the smaller molecules travel faster
  through the agarose. Over time this separates
  the various sized fragments of DNA.
• The gel is then removed and stained for DNA.
  This results in a gel which shows several bands
  of stained DNA.

9
Finished Gel
10
Gel Electrophoresis
11
DNA Fingerprinting
DNA is now a powerful tool in identification. Base
d on the fact that the amount of "junk DNA"
differs uniquely between individuals. Structural
genes are often separated by large regions of
repeating basepairs. The number of these repeats
is unique to an individual. Therefor when DNA
from a person is cut with a restriction enzyme,
the length of the fragments will be unique to an
individual.
12
DNA Fingerprinting Contd

• This will therefor produce a unique banding
  pattern following a gel electrophoresis.
• This test is highly accurate, and the probability
  of another individual possessing an identical
  banding pattern is estimated as around
  114,000,000,000.

13
DNA Fingerprinting
14
Cloning
15
Cloning What it is

• Cloning is the process of making a genetically
  identical organism through nonsexual means. It
  has been used for many years to produce plants
  (even growing a plant from a cutting is a type of
  cloning). Animal cloning has been the subject of
  scientific experiments for years, but garnered
  little attention until the birth of the first
  cloned mammal in 1997, a sheep named Dolly. Since
  Dolly, several scientists have cloned other
  animals, including cows and mice. The recent
  success in cloning animals has sparked fierce
  debates among scientists, politicians and the
  general public about the use and morality of
  cloning plants, animals and possibly humans

Dolly, the first mammal clone
16
Dolly A Mammal Clone

• Dolly
• In 1997, cloning was revolutionized when Ian
  Wilmut and his colleagues at the Roslin Institute
  in Edinburgh, Scotland, successfully cloned a
  sheep named Dolly. Dolly was the first cloned
  mammal.
• Wilmut and his colleagues transplanted a nucleus
  from a mammary gland cell of a Finn Dorsett sheep
  into the enucleated egg of a Scottish blackface
  ewe. The nucleus-egg combination was stimulated
  with electricity to fuse the two and to stimulate
  cell division. The new cell divided and was
  placed in the uterus of a blackface ewe to
  develop. Dolly was born months later.