GHOST-BAND UN-MASKED

BSS032 at BANGOR.AC.UK BSS032 at BANGOR.AC.UK
Thu Apr 14 09:33:13 EST 1994


Dear Ghost-Band-Busters,

A few weeks ago I posted an offer of an enzyme which degrades ssDNA, linear
dsDNA and nicked circles BUT DOES NOT DAMAGE native plasmids (cccDNA or RFI).

NOW for the result. I took 30 ul of buffer (glycine at pH9/KOH, 50 mM, MgCl2
at 10 mM) containing approx. 3 ug of pUC19 (QIAGEN prepped) containing the
famous 'Ghost-Band' (supplied by Dr. Jimmy Thomson, Max-Planck-Inst,Goettingen
Germany). this was split into 3 aliquots and treated with either nothing,
1 ug T5 exonuclease, or 0.01 ug T5 exo. All three were incubated at 37degC
for 20 mins. Then heat killed at 70degC (ortherwise the DNA/protein complex
is gel shifted), then run agarose.


RESULT. The untretaed plasmid prep. contained several bands typical of
QIAGEN prepped DNA i.e. cccDNA, some nicked (relaxed or RFII), some genomic
DNA (running high up the gel) and our 'GHOST" running slightly ahead of the
brightest (native plasmid) band. HOWEVER, the T5 exo treated samples had
both lost their 'GHOSTS', genomic and nicked bands, but retained the strong
ccc (or RFI) plasmid band!!!

We are doing a couple of more experiments to tie this up, BUT we can already
say that the 'GHOST' has nothing to do with phage origins of replication as
some netters suggested.  It seems likely that the band issome form of replicat-
ive intermediate singlestranded DNA. We are checking now but strongly suspect
that it IS circular single-stranded plasmid. Hence it is resistant to restriction enzyme cleavage etc.

Jon R. Sayers *   AND Jimmy Thomson

Jon R Sayers (Cell and Molecular Biology), Biochemistry Dept.,
University College North Wales, Univ of Wales, Bangor, LL57 2UW.
Tel +44 248 38 23 54
Fax +44 248 370731
Email bss032 at uk.ac.bangor



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