new organisms

RYBICKI, ED ED at micro.uct.ac.za
Wed Dec 8 03:43:04 EST 1993


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....

Enjoy,

 
  ____________________________________________________________________
 | 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)             |
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           First Albert, now Frank...go well, axemen!



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