Unit6NucleicAcidExtractionMethods.pptVIP

Unit 6 Nucleic Acid Extraction Methods Terry Kotrla, MS, MT(ASCP)BB Fall 2007 Purpose To release nucleic acid from the cell for use in other procedures Must be free from contamination with protein, carbohydrate, lipids or other nucleic acids. Used pure nucleic acids for testing. Isolation Routinely isolated from human, fungal, bacterial and viral sources. Pretreat to make nucleated cells available, whole blood Tissue samples Microorganisms Need sufficient sample for adequate yield. Organic Isolation Must 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/Chloroform Biphasic emulsion forms Hydrophobic layer on bottom has cell debris. Hydrophilic layer on top has dissolved DNA Remove top layer, add cold ethanol, DNA precipitates out. Inorganic Isolation Methods Also 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). I know this is not scientific but the precipitated out DNA is usually referred to as “snotty”. Solid Phase Isolation More rapid and effective Use solid matrix to bind the DNA. Wash away contaminants. Elute DNA from column Textbook page 70, Figure 4.3 Solid Phase Isolation The diagram below explains the attractive properties of solid phase for DNA and RNA. Crude Lysis Used for: Screening large numbers of samples Isolation of DNA in limited amounts Isolation from challenging samples, ie, paraffin embedded tissue Usually not done in clinical laboratory. Textbook page 71, Figure 4-4 Isolation of Mitochondrial DNA Mitochondrial DNA is passed from generation to generation along the maternal lineage. Centrifugation to separate out Lyse Precipitate with cold ethanol. Isolation of RNA Requires STRICT precautions to avoid sample degradation. RN