Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ.

ABSTRACT: In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the A

SUBMITTER: Mignardi M 

PROVIDER: S-EPMC4678841 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature