Detection of very-rare-copy DNA in 0.2-ml dried human blood blots.
A nested PCR assay for chromosome 7 inversions (as identified by the presence of T-cell receptor trans-rearrangements) can detect as rare a frequency as 1 copy in 300,000 leukocytes. To identify such rare occurrences from dried blood blots, the most conveniently obtained and stored samples for field population studies, demands a DNA extraction method that will provide both high quality and high yield. We have satisfied this requirement by extracting proteins and other components directly from the minced filter with phenol, before extracting the DNA with Chelex-water. This provides a near maximal yield of denatured DNA of sufficient quality to detect these translocations with a sensitivity equivalent to that of DNA purified from whole blood samples. Blots stored 6 months worked as well as fresh blots. In addition, we present a method for obtaining native DNA from the dried blots, although at a much lower yield. The successful use of blood blots to detect such rare events signals the feasibility of large-scale field studies involving diagnostic molecular epidemiology.
Fishbein
W N
WN
Department of Environment Pathology, Armed Forces Institute of Pathology, Washington, DC 20306-6000, USA.
Foellmer
J W
JW
Davis
J I
JI
Kirsch
I R
IR
D002897:Chromosomes, Human, Pair 7; D004247:DNA / Q000097:blood* / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D006801:Humans; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
D004247:DNA