title: Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly.
abstract: We present an approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single-copy sensitivity and single-base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this technique by successfully detecting six single-copy target sites on human mitochondrial and autosomal DNA. We also demonstrate the high selectivity of this method by detecting X- and Y-specific sequences on human sex chromosomes and by simultaneously detecting three sequence-specific target sites. Finally, we discriminate two target sites that differ by 2 nt. The PNA-RCA-FISH approach is a distinctive in situ hybridization method capable of multitarget visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific.
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA.
authors.forename: Anastasia I
authors.initials: AI
authors.lastname: Yaroslavsky
authors.forename: Irina V
authors.initials: IV
authors.lastname: Smolina
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism; D015894:Genome, Human / Q000235:genetics*; D054629:Genome, Mitochondrial / Q000235:genetics; D006801:Humans; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism*; D061848:Optical Imaging / Q000379:methods*; D020135:Peptide Nucleic Acids / Q000235:genetics / Q000378:metabolism*
chemical_list: D004272:DNA, Mitochondrial; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D004247:DNA