> ABRF recipients,
> I spend my days in the protein world and I now find myself reading a paper
> about cDNA preparations and screening. In this paper it states that nested
> deletions of 2 cDNA clones were generated. What does this mean and why are
> the authors doing this? I have looked in the limited molecular biology books
> that I have and not found the answer. Thank you in advance for any help given.
> Kelly Cass-Samodra
Kelly,
I'm going to jump at answering this question because it will be the first time I
can be a dyed in the wool gray beard. Let me put aside my walker and sharpen my
quill .
a cDNA nested deletion: Way back before Lee Hood and others thought of making an
automated DNA synthesizer it was a bit of a pain to sequence a long length of
DNA. Long was anything over 100 nucleotides. Sanger sequencing was just showing
some promise (although finding some usable Klenow as your DNA polymerase was
tough) and everyone thought Maxam and Gilbert would rule forever. So since you
could not "walk" through a gene by making another oligo the only thing to do
would be to make a bunch of subclones of the unknown fragment and sequence each
subclone then assemble. Genome projects do this by digesting with one or more
enzyme and then sequencing many smaller clones to assemble the larger contig.
But I digress: Nested deletions was the clever way people used to create sub
clones for sequencing. Cut the vector once between the sequencing primer site and
the 5' side of the unknown sequence. Exonuclease III would then be used to chew
in the 5' end. At various time points an aliquot wold be taken out and Exo III
quenched . All of the aliquots would then be tossed together with a sinlge
stranded exo to remove the tails. From there it's a few steps to blunt end
ligate,competent cells and voila, lots of clones that have varying amounts of DNA
removed starting at the original cut site. Oops, I think I left something out.
You need someting to get rid of the 5' overhang on the other side of the first
cut or else exo III will merrily chew back into the primer site in the vector
that will be used for sequencing. A second restriction enzyme between the primer
and the first cut site that leaves 3' overhangs will do. Exo III can't do diddly
to that side now and the deletions are now "nested" with the primer site parked
ready to sequence all the clones. Now I remember the absolute bummer part of this
procedure. Since double stranded DNA sequencing had not been invented yet you had
to size these clones ( to get a nice ladder set) after you had grown them up as
single stranded phage. I loved phage but the single stranded DNA would not gather
up enough EtBr to illuminate a gnats ass. Now that we live in a civilized world
and oligos are cheap I would not think this pathway of nested deletions for DNA
sequencing would be worth the trouble because I left out a ton of steps. And the
exo would always stall at one hairpin and you end up sequencing the same 50
nucleotides over and over again because you couldn't read the dim sizing gel.
But I'll take some wild guesses at a reason:
1) They're masochists.
2) They're a genome lab and masochists. (Is that redundant?)
2) They don't want to sequence the DNA but generate nested deletions of the
expressed protein for activity. They would do this on the poly A end of the cDNA
adding a stop codon during the ligation. Naayh.
3) They make N terminal deletions of the expressed protein so that they could
find some use for their Edman sequencer since mass spec put it in moth
balls....THAT WAS A JOKE. DO NOT FLAME ME. I'M KIDDING. JIM LOVES THE 470. WE
WOULDN'T THINK OF MOTHBALLING IT. #3 IS A JOKE. Really.
And to the masochistic DNA sequencers who read this whole thing and sent in one
of the 240 chromatograms into the DNA sequencing study. Thanks.
Laura S weather update: lots of rain but now fair skies and there are little buds
on the trees. Time to oil the rollerskates. (Not blades, I am a grey beard and
blades bump into my walker.)
-Paul
Paul Morrison D830
Molecular Biology Core Facilities
Dana-Farber Cancer Institute
44 Binney Street
Boston, MA 02115
p_morrison@dfci.harvard.edu
http://mbcf.dfci.harvard.edu