Mesoplasma florum:Microarray design

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The complete genome of the organism Mesoplasma florum has been sequenced and is available in Genbank (accession number NC_006055). An array which includes probes for all of the genome of this organism is valuable for several applications:

  • transcription analysis: what genes are being transcribed, and at what relative levels
  • close species hybridization: we have DNA from other strains of the organism and many close species. Hybridization of DNA from these other organisms against the MF genome array will allow determination of genes unique to MF.
  • determination of the inessential gene set with the TRASH technique (Rubin pmid=12657046, pmid=11606763). This approach uses transposon mutagenesis to insert a selectable marker into the bacterial genome. Cultures are grown under selection, requiring the presence of the marker at a random (but non-lethal) location on the genome. Bulk genomic DNA is prepared from this culture. An outwardly oriented T7 promoter within the transposon is used in vitro to create RNA containing regions close to the insertion site. RNA is extracted and hybridized against the array to identify allowable insertion sites.
  • verification of completeness of cloned YAC constructs
  • tracking the deletion process during genome editing and rewriting

Array design:

  • overlapping probes, 25mers
  • spaced every 10 bp on the genome
  • with and without a central mismatch (A or G preferred)
  • in both orientations
  • randomized locations, in duplicate
  • the array design would include probes for the common marker genes, known mycoplasma phages, and insertion elements

With a 793Kb genome, this is 80000 probes x mismatch (2) x direction (2) x locations (2) or 640,000 probes in the array. If this is too many, we can scale down the overlap until it fits.