Southern hybridisation for HTLV-I provirus

Ivan Bastian ivan at menzies.su.edu.au
Thu Dec 2 16:47:53 EST 1993


Dear Netters,

I am having difficulty detecting integrated HTLV-I provirus in human genomic
DNA by Southern hybridisation. We have a patient who probably has a chronic
leukaemia due to this virus therefore about 10% of her circulating
lymphocytes may contain a copy of the provirus integrated at the same site. I
have extracted high MW DNA from her PBMCs and have done the following:

EcoRI digest (10ug genomic DNA with 25 Us EcoRI/buffer/BSA for <3 hrs at 37
C)

1% agarose gel electrophoresis 35V overnight (UV transillumination shows
fully-digested DNA)

short-wave UV exposure for 2 minutes

alkaline blot to Hybond N+ for five hours (rehydration of gel with TAE and
EtBr shows no remaining DNA in gel)

shortwave UV x 2 mins (supposedly fixes DNA to filter and improves
hybridisation); soak in 2 x SSC then bagged and placed in fridge till hybridisation

prehyb in 10 mls of 5X Denhardts/0.5% SDS/6xSSC/500ug/ml herring sperm DNA
for 5hrs at 65 C

random primer label (using Giga-prime kit) 100ng of pUC18 containing a 955bp
insert representing the env gene of HTLV-I (I have also labelled 100ng insert
itself on recent occasions); I have got specific activity of about 10 8
cpm/ug; using alpha 32 P dATP; I add all the labelled probe

hybridise overnight at 65 C shaking in a water bath

wash twice for 1 hour in 1xSSC/0.1%SDS

expose to film for 1-7 days at -70 C

After all of this on several occasions, I have:

(1) not seen any bands, particularly no bands greater than 9kb (EcoRI does
not cut within the HTLV-I genome therefore one expects to see a band greater
than 9kb)
(2) nor have I obtained bands in DNA from cell lines known to be HTLV-I
infected in which every cell would have at least one proviral copy
(3) I have an unusual background where there is low-level background
reactivity above 4-5kb but no background below this level
(4) I have included pico- to nano-gram quantities of the plasmid from which
I've obtained my probe and this is hybridising brilliantly

Am I doing something stupid? Does anyone have any suggestions?

Thank you for replying directly to my e-mail address, as well as the net,
because our net access is not reliable.

Regards

Ivan

Ivan Bastian
Research Officer
Menzies School of Health Research
ivan at menzies.su.edu.au



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