At 02:59 AM 4/23/98 +0900, whatwhy wrote:
>>I want to know the method isolated DNA from parasite in the erythrocyte.
>The method was NOT affected by the condition of blood-storage.
>whatwhy at netsgo.com
Blood and parasites, like most biological materials, have effective
hydrolases for DNA and other molecules, so one should try to work reasonably
fast on reasonably fresh samples.
Processing and storage methods are crucial with regard to recovering
relatively pure parasite DNA with minimal shearing or degredation, with low
amounts of host DNA; remember that protist genomes are about 1,000-10.000x
smaller than host genomes, so even a few remining host white cells will mean
insignificant percentages of Plasmodium or Babesia DNA. On the other hand,
one need not have highly pure or intact DNA for most PCR uses. For making
libraries or other steps that need a lot of fairly pure DNA, it is helpful
to use blood from which white cells are efficiently removed; most simply,
wash blood cells about 6x, removing about the top 5% volume of buffy coat
each time, and start a culture system with these cells, to be harvested near
maximal growth; this gives about 80% parasite DNA, about the same one gets
with two passages of the blood through white cell removal filters; to do
better, cesium banding helps. In terms of storage, Zolg et al (ca 1987)
used a "high salt lysate" procedure with CsTFA and saponin that kept DNA
intact indefinitely, but few seem to have adopted this. PureGene's system
seems to work well for smaller amounts of filter-collected blood.
There are dozens of usable methods for near-quantitative DNA isolation from
Plasmodium in erythrocytes. An old method is to lyse whole blood 4x with
0.5% SDS in Tris-EDTA buffer, pH 8, then digestion with proteinase K
overnight at 55 C, followed by phenol and phenol-chloroform extraction. I
also like IsoQuick guanidinium chloride-based kits for versatility and
recovery. For very rapid processing for PCR, I like GeneReleaser, which
uses only one microliter of blood. For pulsed field electrophoresis, the
sample is embedded directly into agarose and processed without shearing. I
actually haven't found a method that doesn't "work" for isolating parasite
DNA from blood unless the recommended procedure for blood is not followed.
Blood is very concentrated in protein that can coagulate and heme and
anticoagulents that can stops PCR. Related tricks are to dilute blood in
water if the mixture seems very viscous and to do several extractions; to
wash the erythrocytes in citrate buffer (and to collect in
citrate-P-dextrose as an anticoagulent, for larger amounts of blood); and
always use the more rigorous of alternative purification methods. However,
most DNA isolation methods work well.
Gerald McLaughlin, Ph.D.
Dept Pathology & Laboratory Medicine
Indiana University School of Medicine
635 Barnhill Drive, MS A128
Indianapolis, IN 46202-5120
E-mail: gmclaugh at iupui.edu
Ph 317-274-2651; pager 275-5000, I.D. #2916