Southern hybridisation for HTLV-I provirus

Ivan Bastian ivan at
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

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.



Ivan Bastian
Research Officer
Menzies School of Health Research
ivan at

More information about the Methods mailing list