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Isolation of DNAPurposeTo release nucleic acid from the cell for use in other proceduresMust be free from contamination with protein, carbohydrate, lipids or other nucleic acids.Used pure nucleic acids for testing.IsolationRoutinely isolated from human, fungal, bacterial and viral sources.Pretreat to make nucleated cells available,whole bloodTissue samplesMicroorganismsNeed sufficient sample for adequate yield.Organic IsolationMust purify DNA by removing contaminants.Accomplished by using combination of high salt, low pH and an organic mixture of phenol and chloroform.To avoid RNA contamination add RNAse, enzyme that degrades RNA.

Phenol/ChloroformBiphasic emulsion formsHydrophobic layer on bottom has cell debris.Hydrophilic layer on top has dissolved DNARemove top layer, add cold ethanol, DNA precipitates out.Inorganic Isolation MethodsAlso called salting out.Uses low pH and high salt condition to selectively precipitate proteins.DNA is left in solution (picture on far left).Precipitate out DNA with isoproproanol (middle and right side pictures).

Solid Phase IsolationMore rapid and effectiveUse solid matrix to bind the DNA.Wash away contaminants.Elute DNA from column

Solid Phase IsolationThe diagram below explains the attractive properties of solid phase for DNA and RNA.

Crude LysisUsed for:Screening large numbers of samplesIsolation of DNA in limited amountsIsolation from challenging samples, ie, paraffin embedded tissueUsually not done in clinical laboratory.Isolation of Mitochondrial DNAMitochondrial DNA is passed from generation to generation along the maternal lineage. Centrifugation to separate outLysePrecipitate with cold ethanol.

Isolation of RNARequires STRICT precautions to avoid sample degradation.RNA especially labile.

RNAsesRNases are naturally occurring enzymes that degrade RNACommon laboratory contaminant (from bacterial and human sources)Also released from cellular compartments during isolation of RNA from biological samples Can be difficult to inactivateRNAsesRNAses are enzymes which are small proteins that can renature and become active.MUST be eliminated or inactivated BEFORE isolation.CRITICAL to have a separate RNAse free area of lab.Protecting Against RNAseWear gloves at all timesUse RNase-free tubes and pipet tipsUse dedicated, RNase-free, chemicalsPre-treat materials with extended heat (180 C for several hours), wash with DEPC-treated water, NaOH or H2O2Supplement reactions with RNase inhibitors