Fig. 3: Efficiency of 2′-O-methyl RNA primer extension by the T7 RNA polymerase.
From: Simple synthesis of massively parallel RNA microarrays via enzymatic conversion from DNA microarrays
a PAGE analysis of primer extension in solution. Lane 1: a 57 nt reference product oligonucleotide made using conventional solid-phase synthesis and consisting of a 5′-psoralen 27 nt 2′-O-methyl RNA section and a 3′ 30 nt RNA section (arrow A) plus TURBO DNase. Lane 2: 62 nt DNA reference of template and linker made using conventional solid-phase synthesis and crosslinked to the 27 nt 2′-O-methyl RNA primer, followed by DNase degradation. Degradation products are the primer crosslinked to DNA fragments and residual primer (arrow B). Lane 3: product of the full enzymatic conversion and template degradation of the conventionally synthesized and crosslinked oligonucleotides used in lane 2, with arrow C showing the full-length RNA extension of the 2′-O-methyl RNA primer. The steps performed on each sample loaded into lanes 1-3 are illustrated in the scheme below. b The efficiency of on-array primer extension of in situ synthesized DNA is demonstrated using templates with a single dA introduced at every third position within non-dA mixed-base 61 nt sequences. Conversion is indicated by fluorescence of incorporated Cy3-UTP in the RNA strands (85 replicates). Scale bar is 100 µm. Source data are provided as a Source Data file.
