HELP, problem with forensic sample

srgcles at grv.grace.cri.nz srgcles at grv.grace.cri.nz
Wed Aug 18 23:17:47 EST 1993


DEAR NET

PROBLEM WITH FORENSIC SAMPLE

We have a problem with a forensic case.  We have a crucial bloodstain
on a pair of blue trousers from which are trying to get an RFLP DNA profile.

DNA extracted from the stain was very difficult to digest with HinfI. After 
hitting an aliquot of extracted DNA with excess enzyme, we did get digestion,
 but no profile despite reasonable DNA loading.

Subsequently, extraction of an unstained area of the trousers gave what 
appeared to be hi molecular weight DNA after ethidium staining : A distinct 
'DNA' band at approx 20kb with a lighter smear down the lane. We think the 
band at 20kb may be a contaminant of some kind, possibly not even DNA.

Our extraction is pretty standard, with a protocol adopted by the UK forensic 
science service. The extraction is a proteinaseK/SDS digestion on a sample of 
the bloodstain cut from the trousers. We spin the extract thru a hole made in 
the bottom of the eppendorf into a second (unholed) eppendorf. Then the DNA 
is phenol\chloroform extracted once, ethanol precipitated, resuspended in H2O,
dialysed in TNE before measuring its concentration. The digest is done with
standard NEBuffer2, 4mM spermidine, and works a treat with just about every 
other sample like this weve done.

As you can imagine the sample is very precious and we can't afford to waste it.
 
Does anyone out there have any useful ideas? we're getting desparate.

Our two questions are:

1) What is the background material likely to be (what else glows with ethidium
??) and how can we get rid of it?

What is the best approach to get a profile from this (small) bloodstain?

Thanks for your help

Brian Scrimshaw, Steve Cordiner
Institute of Environmental Health & Forensic Sciences
Wellington
New Zealand



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