Extension product capture improves genomic sequencing and DNase I footprinting by ligation-mediated PCR

An improved protocol for genomic sequencing and footprinting by ligation-mediated PCR.

In vivo footprinting requires the precise and sensitive mapping of rucks in highly complex genomic DNA. The Ugation-mediated PCR methodology (LM-PCR (I, 2]) has overcome sensitivity problems due 10 a selective amplification of the DNA fragments under srudy _ 1be currenl procedure (3) employs three genespecific oligooucleotides (pl-P3) to prime three DNA synthesis reactions (Figure I): First, PI...

Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR.

Ligation-mediated polymerase chain reaction (LMPCR) provides adequate sensitivity for nucleotide-level analysis of single-copy genes. Here, we report that chromatin structure can be studied by enzyme treatment of permeabilized cells followed by LMPCR. DNase I treatment of lysolecithin-permeabilized cells was found to give very clear footprints and to show differences between active and inactive...

In vivo footprinting using UV light and ligation-mediated PCR.

DNaseR: DNase I footprinting analysis of DNase-seq data

The combination of DNase I digestion and high-throughput sequencing (DNaseseq) has been used recently to map chromatin accessibility in a given tissue or cell type on a genome-wide scale (Song and Crawford, 2010). In addition to DNase I hypersensitive sites (DHSs), short regions of protected nucleotides known as footprints can be detected using a technique known as ”digital genomic footprinting...

Enhanced genomic sequencing and in vivo footprinting

We have developed a simplified procedure for the ligation-mediated polymerase chain reaction (LMPCR) using Thermococcus litoralis DNA polymerase (Vent DNA polymerase). We show that Vent DNA polymerase produces correct, blunt-ended primer extension products with substantially higher efficiency than Thermus aquaticus (Taq) DNA polymerase or modified T7 DNA polymerase (Sequenase). This difference ...