Biotechnology: Tools and Techniques of the Trade

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Biotechnology Tools and Techniques of the Trade

• What can you do with DNA?
• Chapter 8

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Success Criteria

• At the end of class today, you will be able to
• Explain what a restriction enzyme is and what
  role they play in the cell
• Explain how restriction enzymes have been used by
  biologists as a tool in the manipulation of DNA

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Biotechnology

• biotechnology manipulation of _______________
• To study the functions of individual genes,
  molecular biologists will cut them out of a
  genome and place them into bacteria.

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Basic Techniques

• Six basic techniques will be covered
• DNA isolation
• restriction enzyme digest
• PCR
• gel electrophoresis
• sequencing
• gene cloning

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1. DNA Isolation
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Isolating DNA

• Before DNA can be manipulated, it needs to be
  isolated from the cells.
• Cell membranes are
• DNA precipitation
• DNA isolation / storage

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DNA Isolation
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2. Restriction Enzyme Digest

• pp 366 - 367

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Restriction Enzymes
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Restriction Enzymes

• restriction endonucleases
• recognition site

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Restriction Enzymes

• Why are these enzymes used?
• Where do they come from?
• What role do they have in nature?

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Restriction Enzymes

• Recognition sites.
• Palindromic.
• Which of the following would be palindromic?
• a) ATACTC
• b) AACGTACGTT
• c) CGGCCGGC
• d) ACGCGT

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Restriction Enzymes

• Restriction enzymes are named according to the
  organism from which it was identified.
• Ex. EcoRI
• E - Escherichia
• co - coli
• R - strain RY13
• I - 1st enzyme in this strain

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Restriction Enzymes

• sticky ends
• If the following piece of DNA is cut with Xho1,
  what ends would be created? (See page 367 for
  recognition site)
• 5-ATTCGCTACTCGAGCTCGATCCTCGAGGCGCAATC-3

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Restriction Enzyme Cutting

• blunt ends

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DNA Ligase

• T4 DNA ligase

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Recombinant DNA

• recombinant DNA

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• Recombinant DNA Animation