Biotechnology 2010 - PowerPoint

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 Genetic engineering:     altering
the sequence of DNA
 Ideas established in early 70's
by 2 American researchers,
Stanley Cohen (worked with
plasmids) and Herbert Boyer
(restriction endonucleases)
 Initially had no commercial
applications for their
experiments, but things changed
quickly. In 1976 Boyer cofounded
Genetech, first biotech company
to go public on the stock market.
1978: somatostatin became the first human hormone
produced by this technology.
Techniques are now commonplace in molecular biology labs
Other examples:
   Insulin, 90%+ diabetics are reliant on human insulin
   supplied by bacteria.
   Somatropin, used to treat human growth deficiency, from
   dwarfism, Turner's syndrome, also used for AIDS-
   associated wasting syndrome now
 Biotechnology involves the manipulation of DNA and protein
 Molecular biologists analyze and alter genes and their respective
      –   Genetic screening: scanning for genetic mutations
      –   Gene therapy: the alteration of a genetic sequence in
          an organism to prevent or treat a genetic disorder by
          creating working proteins.
      –   Transgenic plants: inserting genes to provide new
          proteins, giving plants new properties
      –   DNA fingerprinting: analyzing pattern of bands that
          are unique to an individual.
      –   Human Genome Project...
The tools the scientists use are very specific to DNA and its
  The DNA first has to be cut out of the source organism
  The DNA has to be isolated
  DNA can then be introduced into the host DNA
    Recombinant DNA is DNA from one source
    organism being put into the DNA of a host organism.
1) Cutting Out DNA:

  occurring enzymes that act like a pair of molecular scissors to
  cut DNA in a predictable and precise manner, at a specific
  nucleotide sequence called a recognition site .
  Hamilton Smith, John Hopkins University, won the Nobel Prize
  in 1978 for discovering restriction enzymes in bacteria (Hind III).
  He found their main purpose was to cut foreign DNA that tried to
  invade a bacterial cell (i.e. DNA from a virus).
  Restriction enzymes are named according to the bacteria from
  which they originate.
       –   Bam HI is from Bacillus amyloliquefaciens, strain H.
           The I indicates it was the first endonuclease isolated
           from that strain.
Recognition sites are usually:
   –    4 – 8 base pairs in
   –    Palindromic: both
        strands have the same
        sequence when read
        in the 5' to 3' direction.
   –    Table 1 p 279:
The restriction enzyme EcoRI binds to 5'-GAATTC-3' / 3'-CTTAAG-5'

EcoRI finds this recognition site and breaks the phosphodiester bond
between G and A, then it pulls apart the two strands by breaking the
H-bonds between the complementary base pairs.
Produces what are called sticky ends (unpaired nucleotides at each
Other restriction
enzymes like AluI
produce blunt ends, or
ends with no overhang.
Sticky ends are usually
more helpful to
molecular biologists as
they can easily be
joined with other DNA
fragments cut by the
same restriction
enzyme. Blunt ends
are harder to fuse to a
foreign DNA molecule.
A host must protect its own DNA from endonucleases.
  Methylases are enzymes that place a methyl group (CH3) on
  recognition sites which prevents the restriction enzyme from
  cleaving the DNA at that spot.
  Host DNA is methylated, but foreign DNA is not, so it is can be
  cut by the host cell's restriction enzymes.

                   Restriction endonucleases cleave the
                   DNA into smaller fragments
                    Gel electrophoresis is used to isolate the
                   required gene segment from the rest of the
                    The fragments of DNA will be run through
                   a porous agarose gel using electricity.
                        The fragments of DNA are pulled
                        through pores in the gel due to their
                        negative charge.
                        Smaller fragments will move faster
                        than larger because they can fit
                        through the pores better.
                              VIEWING THE GEL

Plasmids are used by biologists to incorporate genes they want
replicated or transcribed/translated in vast amounts in little time into
bacterial cells.
Vector: vehicle used to introduce DNA into a host cell, ie a plasmid or
                                         1. Restriction enzymes are used
                                         to cut out the gene from the
                                         original cell AND to open the
                                         bacterial plasmid.
                                         2. Once the foreign gene is
                                         isolated it can then be inserted
                                         into the plasmid. The plasmid is
                                         now considered recombinant

3. The recombinant DNA is then
  introduced into a bacterial cell.
  Sometimes a host cell must be
  manipulated to take up the foreign
  DNA plasmid.
  Transformation: introduction of
  foreign DNA (usually by plasmid or
  virus) into a bacterial cell.
  Host cell: cell that has taken up
  foreign plasmid or virus and whose
  cellular machinery is being used to
  express the foreign DNA.
  Competent cell: cell that readily
  takes up foreign DNA.
4) Selection and Cloning
1.   Generation of DNA fragments using restriction endonucleases
2.   Construction of a recombinant DNA molecule
3.   Introduction into a host cell
4.   Selection
           Cells that have been successfully transformed must be
           isolated (usually by antibiotic resistance)
           The vectors used for cloning usually carry an antibiotic-
           resistance gene. Growth of colonies on media containing the
           antibiotic indicates successful transformation.
           Colonies are isolated from media and grown in culture to
           produce multiple copies (clones) of the recombinant DNA
           When the bacteria replicates the recombinant DNA plasmid,
           the new gene product will be formed multiple times (ie. the
           gene is cloned).
     PCR – another means of copying DNA in large numbers

stands for Polymerase Chain Reaction, developed in the late
1980's by Kary Mullis; awarded Nobel Prize in Chemistry in
Does not require a plasmid. Therefore when the process is
finished, it is not necessary to remove the plasmid from the
bacteria and the desired gene from the plasmid. The fragment
is copied directly.
Useful for forensic criminal investigations, medical diagnosis,
genetic research. Only small amounts of DNA are needed.
                                PCR Process

PCR is amplification of a DNA sequence by repeated cycles of strand
  separation and replication in the laboratory (DNA photocopying).

1. Strands are separated using
2. DNA primers, synthesized in
the lab, are created to complement
the start of the target area to be
3. Temp is decreased and the
primers anneal
4. Taq polymerase (from bacteria)
creates new strands of target area
5. Sequence is repeated over and
over on each of the new strands
    After about 30 cycles more than 1 billion copies of the targeted area
    will exist (230).


“Restriction fragment length polymorphism”
Entire genome is subjected to restriction
enzyme digestion
DNA run on an agarose gel, using gel
Single stranded DNA transferred to a membrane
ssDNA hybridized with radioactive probes for
specific regions (such as alleles or areas known
as variable number tandem repeats, that lead to
a specific disease).
An X-ray film is developed, called an
autoradiogram, and the pattern can then be
used to identify a suspect, or detect a genetic

                   SEQUENCING DNA

Sanger dideoxy method: uses DNA replication and dideoxy
nucleoside triphosphates to determine the complementary
Developed by Frederick Sanger and colleagues at Cambridge
University in Great Britain in 1977. They used it to sequence
the genome of a bacteriophage (viral DNA) 5386 base pairs
                      Sanger dideoxy method

- Dideoxy nucleosides are
   missing the -OH group on
   carbon 3 and therefore
   inhibit the process of
- Everytime one is added, the
   process stops and only small
   sequences are created.
- These sequences can be run
   on a gel, and since they will
   run from shortest to longest,
   you can actually read the
   sequence by knowing which
   dideoxy nucleoside was used
   and therefore stopped
   replication at each point.
              Fluorescent Detection of Oligonucleotides

The Human Genome Project
  used a similar method, but
  also included fluorescence on
  each dideoxy nucleoside, so
  the A, G, T and C's lit up as
  different colours. A computer
  read the sequence from gel
  electrophoresis. Thousands
  of sequencers worked 24
  hours a day, 7 days a week to
  decipher 3 billion base pairs.

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