OK, so it's the silly season...Xmas is coming, the weather is
warming up, holidays are on the mind, the academic year is all over
bar the shouting...so here's a cautionary tale from the lab, which
should give some ammunition to all those doomsayers who claim all
our tinkering around is going to lead to new and terrible organisms
rising from our drains.
Last year I had three students doing wide-spectrum PCR amplification
projects; one on geminiviruses, one on cucumoviruses, and one on
papillomaviruses. Our anonymous student on HPVs - let's call her
Sam - was doing nested PCR on DNA extracted from biopsies, and
consequently had to work as catrefully as possible, with DNA in
separate rooms from the primers, different pipettes at all stages,
product separated from working stuff, etc. She also cloned the odd
interesting-looking product, but only if it did NOT hybridise well
to HPV 16, 18, 33. It all worked well, she got a putatively new HPV
(which had got into the databases before we got the ms. out), and
went on her way.
About six months later, I had a student from the "Thiobacillus
Research Unit" downstairs - let's call her Ros - come up to see me
with a very perplexing sequence analysis problem. She had cloned
some gene from T ferrooxidans into a coli plasmid, and had sequenced
it, and had got some very interesting homologies back from a BLAST
search at NCBI: basically, she had a +/- 300-bp stretch of homology
with the L1 CP gene of several HPVs right in the middle of her T
ferro sequence. This was amazing, and we speculated wildly about
how maybe the CP was derived from (whatever protein), and how this
was fundamental...of course, as sequences got refined and we did the
full alignment, the3 dreadful truth dawned: the sequence was a
340-bp stretch of HPV 33, corresponding EXACTLY to the sequence
amplified by the nested primers....
How it got into Ros's clone is murky in the extreme. It turns out
that, yes, Sam had borrowed a Boehringer DIG-labelling kit from the
Thiobacilli, so the physical connection was made - however, Sam was
not interested in HPV 33, so there was no clone of the PCR amplimer
to contaminate the nucleotide mix / substrate / box; only amplimer
itself. And how did it get into Ros's cloning mix, and how into her
clone? Sloppy practice, perhaps - but it proved necessary to T-tail
clone our PCR amplimers in all our projects, and the success rate
was not high at the best of times, so how did trace amounts of an
amplimer which needed blunt/T-cloning get into a sticky-end
ligation, and WORK?
A mystery - but sort of predictable, if you extrapolate. After all,
amplimers are a parasitic form of DNA which requires humans in order
to replicate; here is one derived from a highly sucessful virus in
the first place, the result of successful parasitism and
streamlining in getting humans to amplify it in vitro in the second
place, finally getting into E coli and going completely independent.
Just the first reported in what I predict to be a long series of
escaped amplimers over coming months and years....
| Ed Rybicki, PhD | |
| (ed at micro.uct.ac.za) | "Lord, won't you buy me |
| Dept Microbiology | A Mer-ce-des Benz |
| University of Cape Town | My friends all have Porsches |
| | I must make amends..." | |
| Private Bag, Rondebosch | |
| 7700, South Africa | - Janis Joplin |
| fax: 27-21-650 4023 | (Pearl, 1971) |
First Albert, now Frank...go well, axemen!