High salt lysates: a simple method to store blood samples without refrigeration for subsequent use with DNA probes.
Blood specimens to be tested for the presence of Plasmodium falciparum using specific DNA probes can be stored as high salt lysates (HSL) without refrigeration. The lysates are prepared from 100 microliter blood samples by a simple 3-step procedure using 2 volumes of H2O to lyse the erythrocytes (step I), 1 volume of a detergent/EDTA mix to lyse the parasites (step II), followed by the addition of 1 volume cesium trifluoroacetate (CsTFA) (step III). The parasite DNA was found to be undegraded, as shown by the unaltered pattern of repetitive sequences obtained after storage of up to 1 month at 37 degrees C, due to the inhibition of DNA degrading enzymes by the cesium salt. The bulk of protein can be removed from the samples by a 1-step precipitation. The addition of 0.3 volumes of a mixture of ethanol: chloroform: isoamyl alcohol (2.5:1:0.04 v/v) precipitates greater than 90% of the proteins from the lysates, leaving greater than 86% of the parasite DNA in the supernatant. The reduced protein content of the samples, when applied to solid supports, results in an increased signal: background ratio on autoradiograms.
D000818:Animals; D001345:Autoradiography; D001769:Blood / Q000469:parasitology; D001793:Blood Preservation; D004247:DNA / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008288:Malaria / Q000175:diagnosis*; D009693:Nucleic Acid Hybridization; D009994:Osmolar Concentration; D010963:Plasmodium falciparum / Q000235:genetics / Q000302:isolation & purification*; D012091:Repetitive Sequences, Nucleic Acid; D013696:Temperature
D004247:DNA