Molecular Biology

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Molecular Biology

• Materials Methods 1
• Enzymes, Cloning, Genetic Engineering

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Why begin with MM?

• Lab is a major portion of this course, and it
  begins today, using this information
• How we know the design interpretation of
  experiments is a major theme of this course
• To jump-start your ability to understand mol.
  bio. methods used in literature
• text strongly emphasizes experiments
• other lit., esp. primary, for Bio Comm, Sr. Sem.,
  and other courses

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Videos of protocols

• www.jove.com
• Journal of Visualized Experiments
• includes a few methods very similar to some
  we will use this semester heat shock
  transformation of E. coli, and plasmid
  preparation using Qiagen kit

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Weavers text Index to experimental methods,
pages xxi-xxii
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Enzymes tools for molecular biology

• Proteinase digests protein
• e.g. Proteinase K cuts at each serine
• Phosphatase removes phosphate group
• e.g. calf intestinal phosphatase, removes
  phosphate from end of DNA
• Polymerase links single nucleotides together
• e.g. DNA Pol I
• Nuclease digests nucleic acids
• Ribonuclease digests RNA
• e.g. RNAse A purifies DNA from mixed nucleic
  acids

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Endonuclease cuts within DNA polymer
Exonuclease chews its way in from the end of DNA
polymer
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Restriction enzymes endonucleases - made by
bacteria - cut DNA at specific sequences, usually
6bp long, recognition sequences are palindromes
named for genus, species, strain from which
they are isolated
strain R
Escherichia coli
first restriction enzyme isolated from that
strain (Roman numeral)
EcoR I
..G AATTC.. ..CTTAA G..
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Lambda (?) a bacteriophage with a DNA genome
Restriction map shows where each different
restriction enzyme cuts the DNA
enzyme Hind III cuts at these places
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• Ligases
• repair breaks in DNA backbone
• make covalent sugar-phosphate bonds
• often require ATP as a cofactor
• named for the organism from which they are
  isolated
• (e.g. T4 ligase is made by bacteria infected with
• the T4 bacteriophage)

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• Ligases
• repair breaks in DNA backbone
• make covalent sugar-phosphate bonds
• often require ATP as a cofactor
• named for the organism from which they are
  isolated
• (e.g. T4 ligase is made by bacteria infected with
• the T4 bacteriophage)

Hence ligases can put together DNA that you have
cut using a restriction enzyme even if the DNA
pieces come from different sources, different
species, etc!
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Scientific American
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Scientific American
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Genetic engineering of prokaryotes in a nutshell
Note source! Reflects ubiquity of Mol. Bio.
National Geographic
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(No Transcript)
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National Geographic
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Transgenic tobacco shoots on kanamycin / timentin
medium (genetic engineering project by Robert
Rutherford, UMM)
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Herbicide-resistant cotton
Insect-resistant tobacco
Virus-resistant potato
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(No Transcript)
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genetically engineered yeast cells (eukaryote)
on an industrial scale
Scientific American
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genetically engineered mammalian cells
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genetically engineered mammals littermates with
or without growth hormone gene added
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Recombinant Bovine Growth Hormone (rBGH) Bovine
Somatotropin (BST) NOT the cows that
are genetically engineere hormone (protein)
is made in prokaryotes
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Agrinews Sept. 1989
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recombinant human growth hormone availability c
ost safety ! (earlier human growth
hormone isolated from cadavers has resulted,
decades later, in Creuzfeldt- Jacob syndrome
big lawsuits)
National Geographic