15.2: Isolation de l’ADN
DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow specific protocols even for genomic DNA extraction.
Types of genomic DNA extraction methods
The main aim of genomic DNA extraction methods is to separate gDNA from proteins, RNA, and other cell content. It involves four basic steps - 1. Disruption of the cell structure mechanically or using chemicals to obtain the cell lysate 2. Protection of DNA from degradation during processing 3. Separation of the soluble DNA from cell debris 4. Elution of purified DNA.
Most genomic DNA isolation protocols are either solution-based or solid-phase extraction methods. Solution-based methods rely on precipitation and centrifugation steps to separate DNA from other cellular material, followed by organic extraction or "salting out" to separate soluble DNA from cellular proteins. The final DNA precipitation is done using ethanol. In contrast, solid-phase extraction methods use solid support, such as silica or cellulose matrices, to bind DNA, followed by washing and DNA elution from the solid support. It involves centrifugation, vacuum, or magnetic methods to separate the bound DNA from other cellular components.
The choice of gDNA extraction method depends on the type of sample, the number of samples to be processed at once, and the downstream application of the DNA.
