DNA Extraction

Main Steps:

· Cell lysis

· Proteinase

· Isolation of nucleic acid

· Precipitation of nucleic acid

Cell Lysis:

· FFPE:

· Deparaffinization

· Xylene (organic solvent)

· rehydration

· Fresh/Frozen:

· Homogenization of tissue

· Blood:

· Centrifugation and isolation of buffy coat layer

· Hemoglobin inhibits PCR

Proteinase:

· 56 ºC

· FFPE:

· Prolonged proteinase

· Heat also reverses formalin cross-linking between proteins (primarily histones) and nucleic acids

Isolation of nucleic acid:

· Organic (Phenol) extraction:

· Nucleic acids are highly soluble in aqueous solution

· Proteins, lipids, and carbohydrates have hydrophobic and hydrophilic regions

· Some are entirely soluble in organic solutions

· Some are selective for the interface between the organic and aqueous phases

· In pH is acidic, RNA can be selectively extracted

· DNA goes into the organic phase

· High-quality nucleic acids

· Relatively labor-intensive

· Uses hazardous chemicals

· Produces liquid organic waste

Precipitation of nucleic acid:

· Ethanol-salt precipitation:

· Addition of concentrated ethanol and salt

· Ethanol makes the solution hydrophobic

· Salt increases the ionic strength of the solution

· Reduces the repulsion of the negatively-charged sugar-phosphate backbone of the nucleic acid

· Centrifugation to collect the precipitate

· Resuspend in a dilute salt buffer (TE buffer) or water

· Chaotropic salt-silica column extraction

· Sodium iodide (NaI) or guanidinium isothyocyanate (GITC)

· Simple, fast

· Commercial kits

· Adaptable to high-throughput robotic methods

· Widely used

·

Yield:

· FFPE:

· DNA longer than that packaged into a nucleosome (~200 bp) is difficult to recover from FFPE tissue

Quality:

· Electrophoresis gel with EtBr

· High-quality, substantially intact DNA forms a single band close to the well

· Degraded DNA shows a smear

· EtBr is mutagenic

· SYBR green may be a better alternative

References:

· Smith-Zagone MJ, Pulliam JF, Farkas DH. Molecular Pathology Methods. In: Molecular Pathology in Clinical Practice.; 2007:15-40. Available at: http://dx.doi.org/10.1007/978-0-387-33227-7_2 [Accessed September 11, 2009].