Biotechnology and Genetic Engineering PBIO 450550

1
Biotechnology and Genetic EngineeringPBIO 450/550

• Characterization of DNA clones including
• Restriction Enzyme (RE) mapping
• Subcloning
• Southerns
• Northerns
• Westerns
• Hybrid-select translation
• DNA sequencing
• PCR

2
Characterization RE mapping
Predict what would happen with a double digest.
3
Characterization Subcloning

• Refers to the process of cloning smaller pieces
  of a large DNA cloning into another cloning
  vector
• E.g., subcloning the individual EcoRI fragments
  of a partial EcoRI lambda genomic clone into
  plasmid vectors
• Facilitates amplication and analysis of the
  subcloned DNA, including probe preparation

4
Characterization Southern blot hybridization
-transfer of DNA from a gel to a membrane (e.g.,
nitrocellulose, nylon) -developed by Edwin
Southern
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Characterization Northern blot hybridization
X RNA
X
x salt
X RNA
-transfer of RNA from a gel to a membrane (e.g.,
nitrocellulose, nylon) -reveals mRNA size (and
approximate protein size), tissue- and
organ- specific expression, and kinetic patterns
of expression
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Characterization Western blotting
X Protein
Enzyme reaction or
X
React with Antibody X
x Buffer requires electric current
X
-transfer of protein from a gel to a membrane
(e.g., nitrocellulose, nylon) -requires the use
of an electric current to facilitate transfer
7
Animations for Western Blotting

• SDS gel electrophoresis- MCB Chapter 3
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n03000i03010.04o00510
  006100052000530005400056000570005900060000
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  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns0
• Western blotting or Immunoblotting- MCB Chapter 3
• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n03000i03010.05o00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns0

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Characterization Hybrid-select translation
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Characterization DNA Sequencing
The dideoxynucleotide structure (note 3H) is the
key to dideoxy DNA sequencing
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Dideoxy DNA Sequencing Animation- MCB Chapter 9

• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n09000i09010.06o00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns592

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Characterization DNA sequencing
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DNA sequencing
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Automated DNA sequencing
Capillary electrophoresis
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PCR Animation- MCB Chapter 9

• http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
  sp?vcategorys00010n09000i09010.07o00510
  006100052000530005400056000570005900060000
  700007100001000020000300004000050010000200
  00300004000050000600007000080000900010000
  110001200013000140001500016000170001800019
  0002000021000220002300099000ns592

15
The Polymerase Chain Reaction (PCR)

• PCR is a cloning method without a host
• Thermus aquaticus, a hot spring bacterium,
  produces Taq polymerase
• Taq polymerase, unlike E. coli DNA polymerase, is
  not denatured at 95C

16
Chemical synthesis of DNA(oligonucleotide
synthesis using phosphoramidite chemistry)