Re: DNA-nested prose

(no name) ((no email))
Thu, 5 Mar 1998 12:03:35 -0700

>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
>

Paul,

I love the way you explained that. You are truly the "James Joyce" of
molecular biology. Or perhaps the ee cummings?

Skip Vaught
University of Arizona
DNA Sequencing Service