Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.
Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.
Department of Pathology, University of Leeds.
D P
DP
Jackson
F A
FA
Lewis
G R
GR
Taylor
A W
AW
Boylston
P
P
Quirke
D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D019286:Endopeptidase K; D005784:Gene Amplification; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D014066:Palatine Tonsil / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification; D012697:Serine Endopeptidases; D012967:Sodium Dodecyl Sulfate; D014021:Tissue Preservation
D012967:Sodium Dodecyl Sulfate; D012313:RNA; D004247:DNA; D012697:Serine Endopeptidases; D019286:Endopeptidase K