15.2: DNA Isolation and Restriction Enzymes
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.
Restriction enzymes and their uses
Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. The host bacteria protect their own genomic DNA from these enzymes by methylating these sites.
Because different bacterial species produce different restriction enzymes, each enzyme has a unique restriction site and is named after the bacterium of origin. For instance, EcoRI is isolated from the E.coli strain RY13.
When DNA is digested with a particular restriction enzyme, all the fragments produced have the same sequence at their 5’ and 3’ ends. Thus, when a plasmid DNA and an insert are cut with the same restriction enzyme, they have complementary ends that can be easily ligated.