You might want to think about cloning in a bacterial artificial
chromosome (BAC) ; for a reference, see PNAS 89:8794 (1992).
Greg Lennon
Human Genome Center
Lawrence Livermore National Laboratory
> From BIOSCI-REQUEST at net.bio.net Wed Nov 10 23:36:11 1993
> Date: Thu, 11 Nov 1993 18:20:33 +1000
> From: U6065606 at ucsvc.ucs.unimelb.edu.au> Subject: Clone a whole chromosome?
> To: biochrom at net.bio.net> X-Vms-To: IN%"biochrom at net.bio.net"
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>> Dear Chromosome Group,
>> We are investigating a small algal chromsome believed to carry genes
> involved in photosynthesis. The chromosome has an apparent size of
> 95kb in pulsed field gels. Would it be feasible to clone the entire
> chromosome into bacteriophage P1? We would welcome comments on the
> following strategy.
>> If the ends of the 95kb chromosome are typical of eukaryotic
> chromosomes, they will contain telomeres with short 3' overhangs.
> These overhangs could be trimmed by the 3'-5' exonuclease activity of
> the T4 polymerase. Dephosphorylated BamH1 adaptors could then be
> ligated to the ends of the chromosome and the adaptors phosphorylated
> to allow the adaptor/chromosome complex to be ligated into the
> dephosphorylated site in the vector.
>> We have chromosome-specific probes and STS PCR primers to screen for
> inserts.
>> We envisage two possible DNA preps as start points.
>> 1/ Cutting the 95kb band out of a pulsed field gel and using agarase
> to get the chromosome out intact.
>> 2/ Alternatively we could use total high molecular weight genomic
> DNA (>200kb) from the algal cells. The size selection would then be
> impemented by the phage packaging system.
>> Does anybody have any experience, opinions, pitfalls to avoid. Is
> this feasible in your experience. We keep telling ourself that we
> only need one clone, not a comprehensive library.
>> Thanks
>> Geoff McFadden and Paul Gilson
> Plant Cell Biology Research Centre
> University of Melbourne, Parkville 3052 VIC
> Australia
>> _--_|\ email: u6065606 at ucsvc.ucs.unimelb.edu.au> / \ telephone: +61 3 344 5062
> \_.--._/ fax: +61 3 347 1071
> v
>>>>
