Fluorescence-based detection of short DNA sequences under non-denaturing conditions.
The ability of peptide nucleic acid (PNA) to open up duplex DNA in a highly sequence-specific manner makes it possible to detect short DNA sequences on the background of or within genomic DNA under non-denaturing conditions. To do so, chosen marker sites in double-stranded DNA are locally opened by a pair of PNA openers, thus transforming one strand within the target region (20-30 bp) into the single-stranded form. Onto this accessible DNA sequence a circular oligonucleotide probe is assembled, which serves as a template for rolling circle amplification (RCA). Both homogeneous and heterogeneous assay formats are investigated, as are different formats for fluorescence-based amplicon detection. Our recent data with immobilized analytes suggest that marker sequences in plasmid and bacterial chromosomal DNA can be successfully detected.
Smolina
Irina V
IV
Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, 36 Cummington Street, MA 02215, USA.
Kuhn
Heiko
H
Lee
Charles
C
Frank-Kamenetskii
Maxim D
MD
D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D004270:DNA, Circular; D005456:Fluorescent Dyes / Q000737:chemistry*; D016678:Genome; D021141:Nucleic Acid Amplification Techniques; D009838:Oligodeoxyribonucleotides / Q000032:analysis*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D020135:Peptide Nucleic Acids
D004270:DNA, Circular; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D004247:DNA