Isolamento do DNA

The first step is cell lysis, which breaks open cells to release DNA into solution. This is accomplished through physical disruption using equipment like homogenizers or sonicators, combined with chemical treatment using detergents that dissolve the lipid-based cell and nuclear membranes. Once cells are lysed, the DNA content floats freely into the surrounding solution for further processing.

Organic solvents such as phenol and chloroform are commonly used to separate DNA from proteins based on their differing solubility. The sample is vortexed with phenol-chloroform and centrifuged to separate aqueous and organic phases. The DNA-containing aqueous phase at the top is pipetted off, leaving proteins in the organic phase below.

Proteinase K and RNase enzymes are added during or after cell lysis to degrade proteins and RNA respectively, leaving DNA intact. These enzymes selectively break down unwanted cellular molecules, improving DNA purity. This enzymatic degradation works alongside physical and chemical separation methods to remove contaminants from the extracted DNA.

Salt is added to stabilize the negatively charged phosphate groups in the DNA backbone, protecting the molecule during extraction. This ionic stabilization prevents DNA degradation and helps maintain its structural integrity throughout the purification process. Salt also assists in the subsequent precipitation step when alcohol is added.

Ice-cold alcohol, such as ethanol or isopropanol, is added to the sample, causing DNA to form a white, cloudy precipitate. The sample is then centrifuged, causing the DNA precipitate to collect at the bottom as a pellet. This method effectively separates DNA from dissolved salts and other soluble molecules in the aqueous phase.

After precipitation, the DNA pellet is washed by removing the liquid, rinsing in alcohol, and centrifuging again. Following the final wash, the pellet is resuspended in a buffer solution, creating a purified DNA solution ready for use in biotechnology applications like polymerase chain reaction and other molecular techniques.

Centrifugation is used at multiple stages to separate materials by density. After cell lysis, it sediments cell debris to the bottom. During phenol-chloroform separation, it divides aqueous and organic phases. Finally, it collects the DNA precipitate as a pellet and removes wash solutions. This repeated use ensures efficient separation and purification throughout the extraction process.