Clone a whole chromosome?

greg at MENDEL.LLNL.GOV greg at MENDEL.LLNL.GOV
Thu Nov 11 14:21:01 EST 1993


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
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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
> 
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