Human Genomic DNA Isolation for Molecular Genetics Clinical Lab

Human Genomic DNA Isolation
 Key steps:
o Cell lysis
 Disruption of cell membrane
o Separation of DNA from macromolecules
 Inactivation/Inhibition of endogenous nucleases/proteases
 Removal of other proteins (histones, other DNA binding proteins)
o Separation of DNA from small molecules
 Removal of detergents & salts
o Concentration of DNA into smaller volume
 Precipitation of DNA
 Gel Electrophoresis
o One lane of raw purified DNA
o One lane of raw purified DNA + all other ingredients except RE
o One lane of DNA + all ingredients with RE
 Potential Sources of DNA
o Blood (buffy coat)
 From peripheral vein
 Use EDTA (purple) or ACD (yellow) tubes
 Isolate DNA within 96 hrs of blood collection for regular prep
 Do not freeze whole blood
o Tissue
 From CVS sampling, pathology lab, POC
o Cultured Cells
 Can use suspension/fibroblast cultures
 Sourced from cytogenetics lab
 Use trypsin to remove cells from flask/dish




 Phenol/Chloroform Method
o Most common method to remove proteins & isolation of the DNA by precipitation with EtOH
o Cell lysis:
 Proteinase K  Degrades membrane proteins
o Separation of DNA from macromolecules:
 Remove proteins by phenol extraction followed by chloroform extraction
 Phenol denatures proteins
 Approx. 10% of phenol is in aq. Layer
 Chloroform also denatures proteins, remove water from phenol
 Isoamyl alcohol  Reduces foaming & stabilizes interface between organic/aq. Phases
o Conc. of DNA into smaller volume
 Precipitate nucleic acids with -OH in presence of high salt, induces transition in DNA
o Notes: