Isolation of nascent strands by nascent strand capture and release (NSCR). (A) Schematic of the approach in which a mixture of RNA-DNA chimeric nascent strands and similar sized contaminating DNA strands are first 5′ end labeled by addition of a 5′ thiophosphate and chemical modification with biotinylated maleimide. 5′-biotinylated oligonucleotides are then bound to a streptavidin column, and the nascent strand component of the bound molecules are specifically released using RNase I. (B) The efficacy of the method is demonstrated by using known substrates in which a mixture of a DNA fragment (representing contaminating DNA) and an RNA-DNA chimera (representing nascent strands and generated as an amplicon from genomic DNA using synthetic RNA[12 nt]-DNA[20 nt] primers) were mixed, 5′ biotinylated, and separated using the methodology. Lanes: B shows the input ratio; F shows the flow through following binding to the column; W shows mock treated fraction after the final wash; R shows material released by RNase I; and C shows material remaining on the column following RNase I treatment as recovered by alkali treatment. Densotometric tracings for lanes B and R are shown below the gel image.
