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.

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