This is sometimes used to get internal sequence from a large clone without
having to make new primers, when using primers from the vector. A set of
pieces of different length (hopefully) of the cloned insert are removed
and the vector religated and then seqenced using a vector primer. Page 127
of the current Stratagene calalog (no affiliation) has a nice diagram
explaining this better than I can. I have tried this and find it works
okay. The main problem was that the rate of digestion was not linear with
time (I suspect the rate of the exonuclease digestion is somewhat sequence
dependent) so the distance between the new ends sometimes was bigger than
anticipated so overlap for sequence confirmation was not always optimal.
Hope this helps. Judith
----------------------------------------------------------------------------
Judith A. Airey
Dept. Pharmacology/318
University of Nevada, Reno
Reno, NV 89557
Tel: 702 784 4651
Fax: 702 784 1620
On Wed, 4 Mar 1998, Paul Morrison wrote:
> Kelly S Cass-Samodral wrote:
>
> > 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
>
>
>
>