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].