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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