Labeling of unique sequences in double-stranded DNA at sites of vicinal nicks generated by nicking endonucleases

Abstract

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.

Figure 1.

Schematic overview of the method. In this study, mainly probe oligonucleotides with an additional, target-independent 3′-terminal section were employed, leading to 3′-flap DNA upon probe hybridization and ligation. Linear rolling-circle amplification (RCA) was used as a reporter system to fluorescently detect probe incorporation events.

Figure 2.

Vicinal nicks are prerequisite for target labeling. (A) Partial sequences of different dsDNA substrates prepared by assembly PCR. The recognition sequence and cleavage site for NEase Nt.BstNBI present in the selected wild-type and prepared mutant λ-DNA sequences are marked. (B) Probe hybridization and ligation of nicked substrates. PCR amplicons (170 bp, lanes 1) were treated with Nt.BstNBI, leading to doubly nicked wt and singly nicked m1 and m2 fragments (lanes 2). Samples were then incubated with probe P*-45k-3′F for 10 min at 50°C, followed by cooling and incubation with T4 DNA ligase for 1 h at 16°C. Here and below, M denotes a 100-bp DNA ladder (NEB). (C) Removal of excess probe oligonucleotide. Probe-labeled wt DNA sample before and after incubation with RecJ_f, a 5′→ 3′ ssDNA-specific exonuclease.

Figure 3.

Real-time RCA. (A) Measured fluorescence from PNA beacon Flu-Glu-AAGGCTAGGAA-K-K(Dabcyl)-NH₂ plotted against the time of RCA reactions containing 500 (red), 100 (blue), 25 (green) or 5 (gray) fmol wt target or 500 fmol each m1 (black) or m2 target (orange), respectively. Inputs comprised aliquots of samples shown in Figure 2 after RecJ_f treatment. Phi29 DNA polymerase was added at t = 1 min. (B) Plot of the initial speed of the fluorescence increase (v_in) as a function of input wt 3′-flap dsDNA present in RCA reactions.

Figure 4.

Detection of genomic λ-DNA. All steps of the assay were performed in solution with the exception of RCA, which was conducted on glass slides following surface-immobilization of samples. (A) Fluorescent images of signals generated by RCA. (a) λ-DNA sample (10 amol), for which the nicking step was omitted. (b–d) Probe-labeled λ-DNA samples. Spotted DNA amounts were: 0.1 amol (b), 1 amol (c) or 10 amol (d). Scale bar, 50 μm. (B) Partial image of the central spot in panel d, acquired at higher magnification (100×). Scale bar, 20 μm. Inset: enlarged view of an image section with reduced signal density. Scale bar, 2 μm.

Figure 5.

(A) Recognition sequences and cleavage positions of currently available NEases. (B) Possible configurations of NEase sequence recognition motifs at sites with vicinal nicks. Overlaps between these motifs and the cleaved DNA segment are minimal or absent in configuration I, moderate in II and III and extensive in IV.

Figure 6.

Sequence specificity of the assay at elevated ligation temperature. (A) Target sequences present in human herpes virus 6 type B (HHV-6B) at nicking with Nb.BsmI and Nb.BsrDI. (B) Individual or combined labeling of HHV-6B target sites. All three sites are amplified within corresponding PCR products, which differ in length to be distinguished in electrophoresis. Ligation reactions were performed for 1 h at 65°C with 5 U of Ampligase.

Figure 7.

Detection of genomic HHV-6B DNA. Fluorescent images of signals generated by RCA. (a) HHV-6B DNA sample (70 amol), for which the nicking step was omitted. (b) λ-DNA sample (150 amol) treated analogous to the HHV-6B sample shown in row e. (c–e) HHV-6B DNA samples after labeling procedure with probe P*-18k. Spotted DNA amounts were: 0.7 amol (c), 7 amol (d) or 70 amol (e).