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MCT = Molecular Colony Technique

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Developed technique to generate duplicate polony gels **Polony radius decreases as template length increases and as the acrylamide percentage increases. ... – PowerPoint PPT presentation

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Title: MCT = Molecular Colony Technique


1
  • 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

2
  • 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.

3
(No Transcript)
4
  • 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)

5
  • Problems to address
  • RT and PCR efficiency
  • DNA contamination
  • primer/dimer interactions
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