Title: Biotechnology and Genetic Engineering PBIO 450550
1Biotechnology and Genetic EngineeringPBIO 450/550
- Characterization of DNA clones including
- Restriction Enzyme (RE) mapping
- Subcloning
- Southerns
- Northerns
- Westerns
- Hybrid-select translation
- DNA sequencing
- PCR
2Characterization RE mapping
Predict what would happen with a double digest.
3Characterization 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
4Characterization Southern blot hybridization
-transfer of DNA from a gel to a membrane (e.g.,
nitrocellulose, nylon) -developed by Edwin
Southern
5Characterization 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
6Characterization 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
7Animations for Western Blotting
- SDS gel electrophoresis- MCB Chapter 3
- http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
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0002000021000220002300099000ns0 - Western blotting or Immunoblotting- MCB Chapter 3
- http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
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8Characterization Hybrid-select translation
9Characterization DNA Sequencing
The dideoxynucleotide structure (note 3H) is the
key to dideoxy DNA sequencing
10Dideoxy DNA Sequencing Animation- MCB Chapter 9
- http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
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11Characterization DNA sequencing
12DNA sequencing
13Automated DNA sequencing
Capillary electrophoresis
14PCR Animation- MCB Chapter 9
- http//bcs.whfreeman.com/lodish5e/pages/bcs-main.a
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15The 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
16Chemical synthesis of DNA(oligonucleotide
synthesis using phosphoramidite chemistry)