Labeling of unique sequences in double-stranded DNA at sites of vicinal nicks generated by nicking endonucleases.
We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3' terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3'-flap DNA is obtained. The single-stranded terminus in 3'-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic lambda-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.
Kuhn
Heiko
H
Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, MA 02215, USA. hkuhn@bu.edu
Frank-Kamenetskii
Maxim D
MD
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D011088:DNA Ligases; D004279:DNA, Viral / Q000032:analysis / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific; D024202:Electrophoretic Mobility Shift Assay; D004706:Endodeoxyribonucleases; D015654:Herpesvirus 6, Human / Q000235:genetics; D008856:Microscopy, Fluorescence; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D004706:Endodeoxyribonucleases; C053864:endodeoxyribonuclease BsmI; D015252:Deoxyribonucleases, Type II Site-Specific; D011088:DNA Ligases