SHOTGUN SEQUENCING

Bruce Roe broe at aardvark.ucs.uoknor.edu
Mon Dec 13 08:30:00 EST 1993


In article <1993Dec7.084144.4413 at crc.ac.uk>, mjones at crc.ac.uk (Dr. M.D. Jones) writes...
>Hi netters
> 
>One of the favoured ways of sequencing large DNA fragments is to shotgun clone 'sonicated'
>DNA into a M13 vector and sequence the clones, and compile the data by computer.
> 
>In the past I have successfully used this strategy, but the most frustrating steps were getting
>enough recombinant clones to complete the sequence.
> 
>This is due to the damage caused by sonicating the DNA, making most of it unclonable.
> 
>Does anybody out there have a detailed fool-proof 'sonication' protocol ?
> 
>Anybody have any advice/tips/etc on how to get the best out of this procedure ?
> 
>On a similar note, does anybody use 'partial' restriction enzyme digestion to
>fragment their target DNA ?
> 
>Fitzgerald et al, 1992 (Nucleic Acids Res., 20(14), 3753-3762) used the enzyme
>CviJI (recognition sequence RGCY) to create essentially random shotgun DNA for
>sequencing.
> 
>Does anybody know if this enzyme is commercially available, particularly in the UK ?
> 
>Many thanks
> 
> 
>Mick Jones
> 
>Department of Virology, RPMS
>Du Cane Road, W12 0NN, UK
>(voice) 081-740-3328
>email: mjones at rpms.ac.uk
> 

Sonciation indeed (as a later poster stated) is a difficult method to
optimize.  The major problems is that the breaks are single stranded
and thus you'll get long ss ends that are difficult to fillin or otherwise
make blunt ended.  To make the ss breaks as close together as possible,
keep the sample as cold as possible.  The things we have done are to use
the cup-horn sonicator in the cold room, replace the ice/water mixture
often and do a series of short bursts rather than long bursts.  We actually
now no longer use sonication but use a nebulizer to generate random fragments
for shotgun cloning.

The protocols we presently use are being published in an upcoming book BUT
the entire article is available on our Gopher site: aardvark.ucs.uoknor.edu

 -->  3.   > Other U of Oklahoma Gophers, Services & Data /

 -->  6.  Oklahoma University Genetic Computer Group (OUGCG)/

 -->  6.  shotgun_sit.hqx [14-Sep 18:22:52, 35KB].

If you don't have Gopher available or don't know about Gopher, then contact
your local computer guru and get them to do something about it.

Here's a copy of the sonication protocol from the manuscript:

Production of random DNA fragments by sonication

	The preparation of random DNA fragments by sonication was performed in
a Heat Systems Ultrasonics W-375 cup horn sonicator (Farmingdale, NY) filled
with ice cold water in a cold room.  It is imperative that the temperature
of the sample and the water in the cup horn sonicator be maintained as close to
0oC as possible throughout the following manipulations to prevent DNA melting
which can result in an uneven fragment distribution pattern.  In preparation
for sonication, a tube holder was prepared by removing the upper two-thirds of
a 50 ml polypropylene centrifuge tube with a conical bottom and cutting a hole
in the bottom to fit the 1.5 ml snap cap conical tube containing the DNA
sample. A solution containing approximately 100 mg of plasmid or cosmid DNA in
350 ml of Tris-magnesium buffer (50 mM Tris-HCl, pH 8 containing 15 mM MgCl2)
was prepared and distributed equally to ten 1.5 ml conical snap cap tubes
stored in an ice water bath.  After the sonication horn was filled with ice
cold water, a snap cap tube containing 35 ml of sample was positioned in the
tube holder, was centered approximately 1 mm above the sonication horn, and was
sonicated for 5 seconds with the sonicator set on 'HOLD', 'CONTINUOUS' and
maximum 'OUTPUT CONTROL' = 10.  The tube then was removed from the sonication
horn to an ice water bath, the ice cold water was replaced, and another sample
was positioned in the cup horn.  This second sample was sonicated for 10
seconds, the third for 20 seconds, the fourth for 30 seconds and the fifth for
40 seconds with each sample being placed in an ice water bath after sonication. 
The ice cold water in the cup horn was replaced between each sample.  After a
brief centrifugation to collect any condensate produced during sonication, 10
ml aliquots of each sonication time point were electrophoresed on a 1% agarose
gel to determine which conditions produced fragments in the desirable size
range of 1-2 kb. Alternatively, fragments in the desirable size range also
could be obtained by a series of individual 10 second bursts, with replacement
of the ice cold water in the cup horn between each burst.  Once the sonication
conditions were optimized for a given DNA sample, the remaining 5
microcentrifuge tubes containing 35 ml aliqouts were sonicated under the
optimum conditions just determined. Subsequently, each sample was centrifuged
to collect any condensate produced during sonication and stored in an ice water
bath before proceeding with the end repair step.

Cheers......bruce
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