MCT = Molecular Colony Technique

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• MCT Molecular Colony Technique
• Alexander Chetverin
• Institute of Protein Research of the Russian
  Academy of Sciences
• References
• NAR(10)2349 from 1993
• AnalBiochem(334)376
• NAR(21)2349
• BioTechniques(33)150
• Polony Polymerase or PCR Colony Technology
• George Church
• Department of Genetics at Harvard Medical School
• References
• NAR(27)e34 from 1999 Methodology
• Science(301)836 Pre-mRNA splicing
• PNAS(100)5926 Genotyping
• AnalBiochem(320)55 FISSEQ

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• Technique
• Acrylamide polymerized in a solution containing
  standard PCR reagents (primers,
  template, dNTPs, enzyme, buffer).
• Up to 360 DNA/RNA molecules of starting template
  per reaction
• Glass microscope slide (treated with
  bind-silane)
  
• 2 x 65 ul frame sealed chambers
  
  (15 mm x 15 mm)
• Run PCR in 16/16 twin tower block
• Visualize by staining with SYBR Green I
  
  and image with Phosphorimager
• Developed technique to generate
  
  duplicate polony gels
• Polony radius decreases as template length
  increases and as the acrylamide percentage
  increases.

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• Applications
• Parallel In situ Sequencing
• FISSEQ (Fluorescent In Situ SEQuencing)
• Relative Expression of Splice Variants
• Allelic Variation
• Chemical Reactions between single RNA molecules
• Molecular Diagnostics (human blood, detecting
  infections)

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• Problems to address
• RT and PCR efficiency
• DNA contamination
• primer/dimer interactions