Genomic Sequencing by Template Purification: Principles and Mapping of Protein-Bound and Single-Stranded Sequences in Vivo

Abstract

A new genomic sequencing procedure is described. This technique involves as a key step the purification of specific sequences from the bulk of the genomic DNA using a biotinylated RNA probe. The specific sequences purified from genomic material are then a suitable template for primer extension with a highly radiolabeled probe or for ligation-mediated polymerase chain reaction. This methodology allows the identification of stable DNA-protein complexes on genomic DNA as well as the mapping of genomic sequences presenting an altered DNA conformation. We present here details of the procedure and some particular aspects that permit the mapping of transcription-associated melted regions of DNA in whole cells and nuclei.