DNA Extraction

 

Main Steps:

ˇ         Cell lysis

ˇ         Proteinase

ˇ         Isolation of nucleic acid

ˇ         Precipitation of nucleic acid

 

Cell Lysis:

ˇ         FFPE:

ˇ         Deparaffinization

ˇ         Xylene (organic solvent)

ˇ         rehydration

ˇ         Fresh/Frozen:

ˇ         Homogenization of tissue

ˇ         Blood:

ˇ         Centrifugation and isolation of buffy coat layer

ˇ         Hemoglobin inhibits PCR

 

Proteinase:

ˇ         56 ēC

ˇ         FFPE:

ˇ         Prolonged proteinase

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

 

Isolation of nucleic acid:

ˇ         Organic (Phenol) extraction:

ˇ         Nucleic acids are highly soluble in aqueous solution

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

ˇ         Some are entirely soluble in organic solutions

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

ˇ         In pH is acidic, RNA can be selectively extracted

ˇ         DNA goes into the organic phase

ˇ         High-quality nucleic acids

ˇ         Relatively labor-intensive

ˇ         Uses hazardous chemicals

ˇ         Produces liquid organic waste

 

Precipitation of nucleic acid:

ˇ         Ethanol-salt precipitation:

ˇ         Addition of concentrated ethanol and salt

ˇ         Ethanol makes the solution hydrophobic

ˇ         Salt increases the ionic strength of the solution

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

ˇ         Centrifugation to collect the precipitate

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

ˇ         Chaotropic salt-silica column extraction

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

ˇ         Simple, fast

ˇ         Commercial kits

ˇ         Adaptable to high-throughput robotic methods

ˇ         Widely used

ˇ          

 

Yield:

ˇ         FFPE:

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

 

Quality:

ˇ         Electrophoresis gel with EtBr

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

ˇ         Degraded DNA shows a smear

ˇ         EtBr is mutagenic

ˇ         SYBR green may be a better alternative

 

 

References:

ˇ         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].