EDTA/Heparin & DNA stability

David MacHugh dmachugh at mail.tcd.ie
Wed Feb 4 07:09:27 EST 1998


Hi,

We are currently looking at mtDNA variation in cheetahs sampled from
various zoos around Europe and Africa.  We have about 50 samples.  All of
these samples come from vacutainers which have been stored in a fridge
since 1994 and in some cases since 1993.  18 of the 50 blood samples have
been stored in vacutainers which have 15% K3EDTA as a preservative and the
rest have lithium heparin.  Because the samples were so old we did
essentially an ancient DNA-type extraction. We used a whole-blood
digestion buffer, A three-step phenol, phenol/CIA, CIA organic extraction
followed by a purification using Boerhinger-Mannheim High-Pure columns
normally used for cleaning up PCR products.  Anyway we found that the 18
samples stored at 4C in EDTA gave DNA yields approx 100-fold more than
those samples stored in lithium-heparin.  This was reflected in the PCR
amplifications also.  The EDTA-stored samples gave beautiful mtDNA PCR
products, while the lithium-heparin gave weak bands and in some cases
failed to amplify at all.

Has anyone else found EDTA to be a much better long-term storage medium
for blood in this way? Also does anyone have any suggestions as to how the
lithium-heparin samples may be retrieved more efficiently?


Thanks in advance.

David.


David MacHugh Ph.D.,
Genetics Department,
Trinity College,
Dublin 2.
Ireland.
Phone:   353-1-608-2719
Fax:     353-1-679-8558
email:   dmachugh at tcd.ie



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