The Case of the Vanishing Intron
nouspike at cmu.unige.ch
Thu Nov 10 09:07:44 EST 1994
Can anybody explain the folowing mystery ?
- When studying my favorite gene in a patient, I discovered an aberrant
plicing phenomenon, due to a cryptic acceptor site, 55nt downstream of the
- When PCRing cDNA in that region of the gene (RT-PCR), I got the expected
290bp band plus a non-stoichiometric amount of shorter (misspliced) product
- When PCRing genomic DNA , I got a single 605bp band.
Sequencing revealled there was a 315bp intron in it, with a poor acceptor
site, explaining why a cryptic donor site is occasionnaly used.
No problems so far...
I recently repeated this analysis with two new patients. In both cases RT-PCR
yielded a 290bp band and a small amount of 235bp misspliced product (as
BUT with genomic PCR I got:
- Two bands for patient #1: the expected 605bp band and ~290bp band !!
- A single short band (~290bp) for patient #2 !!!
Where did the intron go ??????
- The intron is absent in one allele of patient #1, and in both alleles of #2.
BUT, if there is no intron why do I get aberrant splicing in patient #2 ?
- My PCR reaction has been contaminated
by RNA (but it wouldn't serve as a template without previous RT, would it ?)
by cDNA from the parallele RT-PCR reaction.
BUT this does not explain why I don't see the regular 605bp genomic band in #2.
- One of the PCR primer can anneal at a wrong site and this just happens to
yield a product with the same size than the cDNA product.
BUT, not very likely, and why does this happens a) always in #2, b) half the
time in #1, c) never in the original patient ?
I'm sure there is an obvious solution to this mystery, I just can't figure it
out. If you have an explanation, please let me know. Any suggestion is
Thierry Nouspikel, MD ¦ "Un technocrate c'est un mec,
Genetics & Microbiology Dpmt ¦ tu lui poses une question,
University of Geneva, Switzerland ¦ quand il a fini de répondre
nouspike at cmu.unige.ch ¦ tu comprends plus ta question."
fax:41/22/329.34.04 ¦ Michel Colucci, dit "Coluche"
More information about the Methods