Method in Molecular Biology : DNA isolation

In this page I am going to show  DNA isolation  technique which indispensable step in molecular biology. Molecular biologist routinely perform DNA extraction to remove its contaminants such as protein, lipid, and cell wall component, prior to subsequent experiments. They are in need of pure DNA for several applications, such as polymerase chain reaction, restriction digest, molecular cloning, etc. The critical points of those applications is the purity of DNA samples. Contaminants 

There are three types of DNA, each of them need several modification steps to isolate. Genomic DNA (gDNA) is a kind of DNA that compose the entire genome of the cells or organisms. gDNA reside in the nucleus in the form of chromosome in eucaryote, or in the cytoplasm in the bacteria or procaryote. The second type is a plasmid DNA, and the third is mitochondria or chloroplast DNA.

The principle of DNA extraction is based on the three steps. (1) cell lysis, (2) cell digestion, (3) DNA precipitation and purification. The first step is lysis of cell by disrupting membrane or cell wall to release all of the cell contents. There are so many strategies to perform cell lysis which depend on the cell type. Non ionic surfactant like sodium dodecyl sulphonate (SDS) and EDTA usually are used to perform this task. EDTA will bind with magnesium ions and makes cell more sensitive to disruption. EDTA also prevents DNAse to degrade DNA by chelating Mg2+ ion. SDS interacts with membrane phospholipid, so the cell membrane will break and lysis. Cell disruption is a first key step to accomplish DNA isolation. Several modifications are used according to sample type. If we isolate DNA from animal cell or tissue, we just need SDS, EDTA and proteinase K. But if we want to isolate DNA from bacteria, we use lysozyme (gram negative or E coli) or lysostaphin (for Staphylococcus spp) in order to break the cell wall. Several commercial kits using guanidin isothiocyanate to make cell lysis also.

The next step to perform DNA isolation is cell digestion, in this step we are digesting protein and RNA, and then extracting our DNA by using organic extraction or spin column. Proteinase K degrade protein and RNAse A will degrade all the remaining RNA. After cell digestion, we have to separate DNA from cell debriss. Organic extraction method by using phenol:chloroform:isoamyl alcohol is the most used in conventionally manner, but it more laborious and need several time to accomplish this. Most commercial kits use spin column method, in this method we use silica membrane to trap our DNA instead the other cell debriss are filtered through spin collumn during centrifugation steps. Washing DNA is needed to make purified DNA with the lowest impurities such as protein. DNA washing performed by using wash buffer containinng ethanol 70%. After that, DNA from spin collumn are eluated using elution buffer or steril nuclease free water.

Here I provide some DNA extraction protocols from several commercial kits and using organic extraction from Sambrok.

A. Genomic DNA isolation from blood, tissue, and cell culture using Purelink Genomic DNA Kit

B. Plasmid DNA isolation using GeneJet Miniprep Plasmid Kit Thermo Scientific.