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12.S: Techniques of Molecular Genetics (Summary)
Opening the book…
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.