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

Kuhn H, Frank-Kamenetskii MD - Nucleic Acids Res. (2008)

Bottom Line: 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.The method can find applications in sensitive and specific detection of viral duplex DNA.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, MA 02215, USA. hkuhn@bu.edu

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.

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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.
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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.

Mentions: To investigate the exemplary sequence specificity of probe labeling, we selected three target sites present in human herpes virus 6 type B (HHV-6B) generated by NEases Nb.BsmI and Nb.BrsDI (Figure 6A). These sites, representing intermediate configuration III, constitute a good test for our assay since they are nearly identical in length and contain the same 5-bp-long sequence at the 5′-terminus of the cleaved segment. By PCR, dsDNA fragments of different length carrying each one centrally located target site were prepared. Incubation of each fragment with Nb.BsmI and Nb.BrsDI resulted in the formation of doubly nicked DNA molecules in essentially quantitative yield (Supplementary Figure 2A). To correctly assess the sequence specificity in labeling reactions with pooled amplicons, we first generated deliberately all possible incorrect ligation products by performing ligation with T4 DNA ligase under low fidelity conditions. We also prepared corresponding gapped DNA fragments (41), which may be formed in probe labeling reactions to some extent, if dissociated cleaved segments do not rehybridize and if incorrect probes are not ligated into target sites. The various products were then analyzed concurrently with correct ligation products. Under the employed gel electrophoretic conditions, gapped DNA had a higher electrophoretic mobility and incorrect ligation products had markedly reduced mobility in comparison with the three correct ligation products (Supplementary Figure 2B).Figure 6.


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

Kuhn H, Frank-Kamenetskii MD - Nucleic Acids Res. (2008)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2373456&req=5

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.
Mentions: To investigate the exemplary sequence specificity of probe labeling, we selected three target sites present in human herpes virus 6 type B (HHV-6B) generated by NEases Nb.BsmI and Nb.BrsDI (Figure 6A). These sites, representing intermediate configuration III, constitute a good test for our assay since they are nearly identical in length and contain the same 5-bp-long sequence at the 5′-terminus of the cleaved segment. By PCR, dsDNA fragments of different length carrying each one centrally located target site were prepared. Incubation of each fragment with Nb.BsmI and Nb.BrsDI resulted in the formation of doubly nicked DNA molecules in essentially quantitative yield (Supplementary Figure 2A). To correctly assess the sequence specificity in labeling reactions with pooled amplicons, we first generated deliberately all possible incorrect ligation products by performing ligation with T4 DNA ligase under low fidelity conditions. We also prepared corresponding gapped DNA fragments (41), which may be formed in probe labeling reactions to some extent, if dissociated cleaved segments do not rehybridize and if incorrect probes are not ligated into target sites. The various products were then analyzed concurrently with correct ligation products. Under the employed gel electrophoretic conditions, gapped DNA had a higher electrophoretic mobility and incorrect ligation products had markedly reduced mobility in comparison with the three correct ligation products (Supplementary Figure 2B).Figure 6.

Bottom Line: 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.The method can find applications in sensitive and specific detection of viral duplex DNA.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, MA 02215, USA. hkuhn@bu.edu

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.

Show MeSH
Related in: MedlinePlus