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