Clone a whole chromosome?

U6065606 at ucsvc.ucs.unimelb.edu.au U6065606 at ucsvc.ucs.unimelb.edu.au
Thu Nov 11 03:20:33 EST 1993


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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