12.S: Techniques of Molecular Genetics (Summary)
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- Ying Liu
- City College of San Francisco
- Molecular biology involves the isolation and analysis of DNA and other macromolecules
- Isolation of total genomic DNA involves separating DNA from protein and other cellular components, for example by ethanol precipitation of DNA.
- PCR can be used as part of a sensitive method to detect the presence of a particular DNA sequence
- PCR can also be used as part of a method to isolate and prepare large quantities of a particular DNA sequence
- Restriction enzymes are natural endonucleases used in molecular biology to cut DNA sequences at specific sites.
- DNA fragments with compatible ends can be joined together through ligation.If the ligation produces a sequence not found in nature, the molecule is said to be recombinant.
- Transformation is the introduction of DNA (usually recombinant plasmids) into bacteria.
- Cloning of genes in E. coli is a common technique in molecular biology, since it allows large quantities of a DNA for gene to made, which allows further analysis or manipulation
- Cloning can also be used to produce useful proteins, such as insulin, in microbes.
- Southern blotting can be used to detect the presence of any sequence that matches a probe, within a mixture of DNA (such as total genomic DNA).
- The stringency of hybridization in blotting and in PCR is dependent on physical parameters such as temperature and washing solution content.
Key Terms:
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macromolecules lysis detergent chelating agent EDTA nuclease pellet PCR primer thermalcycle denature anneal extend Taq DNApol electrophoretic gel restriction endonuclease EcoRI sticky end blunt end compatible end |
ligation ligase plasmid transformation competent electroporation selectable marker agarose vector band ethidium bromide Southern blot membrane denaturation hybridization washing probe stringency northern blot western blot transgene |
GMO transformation transfection naked DNA vector electroporation microinjection vesicles callus knock-out recalcitrant Agrobacterium particle bombardment T-DNA Ti plasmid position effects stem cells |