title: 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 ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.
authors.affiliation: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-17121 Sweden.
authors.forename: Marco
authors.initials: M
authors.lastname: Mignardi
authors.affiliation: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-17121 Sweden.
authors.forename: Anja
authors.initials: A
authors.lastname: Mezger
authors.affiliation: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-17121 Sweden.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Qian
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, SE-75185, Sweden.
authors.forename: Linnea
authors.initials: L
authors.lastname: La Fleur
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, SE-75185, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Botling
authors.affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, SE-75185, Sweden.
authors.forename: Chatarina
authors.initials: C
authors.lastname: Larsson
authors.affiliation: Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, SE-17121 Sweden mats.nilsson@scilifelab.se.
authors.forename: Mats
authors.initials: M
authors.lastname: Nilsson
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000737:chemistry; D004273:DNA, Neoplasm / Q000737:chemistry; D006801:Humans; D051379:Mice; D009841:Oligonucleotides; D017354:Point Mutation; D012333:RNA, Messenger / Q000737:chemistry
chemical_list: D004272:DNA, Mitochondrial; D004273:DNA, Neoplasm; D009841:Oligonucleotides; D012333:RNA, Messenger