Isolation of DNA
Molecular Diagnostics
(MDx) Review 548
STEP EXPLANATION
Isolation of nucleated cells (if necessary)
Release of DNA from cells
Separation of DNA from cellular debris,
protein, lipids
Concentration
Storage
Centrifugation of whole blood to obtain buffy coat, differential lysis with water or hypotonic
solution to separate WBCs from RBCs, density gradient centrifugation in Ficoll to obtain
mononuclear cells (settle below plasma, above polys/RBCs).
Cells ruptured by mechanical or chemical methods.
Liquid-phase extraction: use of solvents. Multiple manual steps. Limited sample throughput.
Phenol & chloroform are hazardous.
Solid-phase extraction: DNA adsorption onto silica on membrane filters or magnetic particles.
More commonly used. Easier, can be automated.
Precipitation in ethanol or isopropanol in high salt concentration. Resuspension in H 2 O or TE
buffer. Handle with care to avoid breaking DNA.
Extracted DNA can be stored at RT for several months, refrigerated for 1 yr, or frozen at
–20°C to –70°C for up to 10 yr.
