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Biotechnology and Genetic Engineering PBIO 450550

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Refers to the process of cloning smaller pieces of a ... SDS gel electrophoresis- MCB Chapter 3 ... Capillary electrophoresis. PCR Animation- MCB Chapter 9 ... – PowerPoint PPT presentation

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Title: 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
5
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
6
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
    700007100001000020000300004000050010000200
    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

8
Characterization Hybrid-select translation
9
Characterization DNA Sequencing
The dideoxynucleotide structure (note 3H) is the
key to dideoxy DNA sequencing
10
Dideoxy DNA Sequencing Animation- MCB Chapter 9
  • http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
    sp?vcategorys00010n09000i09010.06o00510
    006100052000530005400056000570005900060000
    700007100001000020000300004000050010000200
    00300004000050000600007000080000900010000
    110001200013000140001500016000170001800019
    0002000021000220002300099000ns592

11
Characterization DNA sequencing
12
DNA sequencing
13
Automated DNA sequencing
Capillary electrophoresis
14
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)
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