Identification of distinct template and product exit tunnels allows proposal of a detailed model for template-directed replication with minimal disruption to the circularised RNP.
Based on the L1750 structure, we propose a model for RNA replication in which there are clearly separated exit tunnels for the single-stranded template and product.
As discussed below, the arrangement of the tunnels in LACV L protein suggests an elegant strategy for RNA synthesis whereby the polymerase forces separation of the template and product strands and directs each down distinct exit channels on opposite sides of the molecule.
Diagnostic PCR was performed using 1 μl of genomic DNA as template, and PCR products were digested using AfeI to identify edited products (Supplementary Figure 2f).
The LAMP reaction was optimised at 63 °C for 1 h using bacterial genomic DNA as the template and the products were visualised under ultra-violet light and analysed by agarose gel electrophoresis.
Sequences surrounding RDD sites were PCR amplified using genomic DNA or cDNA as the template, and PCR products were sequenced.
RT-PCR was conducted using DAOY RNA as a template and PCR products were subcloned in TOPO PCR cloning vectors (Invitrogen, Carlsbad, CA) and sequenced prior to use.
DNA fragments were amplified by standard PCR, using 10 ng of genomic template, and the products were analyzed by dHPLC at a minimum of two different temperatures.
The CYP21A1P-specific products were amplified by 5′- and 3′-nested PCR from AS ASLR-PCR product as template, and CYP21A2-specific products were generated from the product of C4B_F-TNXB_R (B-T) primer pair.
The EGFP cDNA was PCR-amplified using EGFP-N1 (Clontech) as a template, and the product was cloned into pcDNA3.1/V5-His TOPO (Invitrogen) to yield pCMV- EGFP (pox).
