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A novel SERRS sandwich-hybridization assay to detect specific DNA target.

Feuillie C, Merheb MM, Gillet B, Montagnac G, Daniel I, Hänni C - PLoS ONE (2011)

Bottom Line: In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA.In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors.As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Géologie de Lyon-Terre Planètes Environnement, ENS Lyon, Université Lyon 1, CNRS, Ecole Normale Supérieure de Lyon, Lyon, France.

ABSTRACT
In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

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The sandwich-DNA hybridization assay principle.(A) Capture and detection probes are added to the solution of target DNA. (B) After the denaturation step (99°C, 10 minutes), the hybridization of the probes to the DNA target is achieved under gentle agitation (55°C, 3 hours). (C) Streptavidin-coated magnetic micro beads are added to the solution (gentle agitation for 30 minutes at room temperature). (D) Hybridized duplexes are captured by a magnet, unbound compounds are washed out. (E) Hybridized detection probes are eluted (95°C, 20 minutes) and are detected by SERRS in proportion of the initial concentration of target.
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pone-0017847-g001: The sandwich-DNA hybridization assay principle.(A) Capture and detection probes are added to the solution of target DNA. (B) After the denaturation step (99°C, 10 minutes), the hybridization of the probes to the DNA target is achieved under gentle agitation (55°C, 3 hours). (C) Streptavidin-coated magnetic micro beads are added to the solution (gentle agitation for 30 minutes at room temperature). (D) Hybridized duplexes are captured by a magnet, unbound compounds are washed out. (E) Hybridized detection probes are eluted (95°C, 20 minutes) and are detected by SERRS in proportion of the initial concentration of target.

Mentions: In this study, we have applied a Surface Enhanced Resonance Raman Scattering (SERRS) approach as an alternative technology to PCR amplification for the specific detection of DNA. SERRS is a vibrational spectroscopy technique whereby the Raman signal of the compound of interest can be amplified up to 1014 fold [23], [24]. SERRS-active molecules possess a chromophore with an absorption frequency close to the excitation frequency, and can adsorb on rough metallic surfaces such as colloidal silver nanoparticles. This adsorption has a doubly positive effect on the Raman signal: (i) it quenches the fluorescence that allows the highly specific Raman fingerprint of the molecule to be detected, (ii) it amplifies the Raman signal. Potential applications of SERRS detection have been under development since 1997 with a view of detecting DNA [25], thus becoming a rapidly emerging field [26]. Our present SERRS sandwich-hybridization assay is based on the specific hybridization of two nucleic probes to target DNA to be detected in solution (Figure 1). The nucleic probe labeled with rhodamine 6G (detection probe) allows the SERRS detection. The second probe, coupled with biotin (capture probe) allows immobilization and purification of the resulting hybridized complex (i.e. target DNA, capture and detection probes). Previous studies have demonstrated that SERRS-labeled synthetic DNA could be detected [25], [27], and that SERRS signal is stable after hybridization of a labeled oligonucleotide probe with a target DNA [26]. The sensitivity of SERRS makes it a valuable alternative non-enzymatic tool to detect DNA. We have developed a study model in which the target and control DNA are homologous sequences of chamois (Rupicapra rupicapra) and common goat (Capra hircus, a closely related species to R. rupicapra), respectively. The results of this new assay shows that DNA of R. rupicapra can be specifically discriminated from C. hircus at the 10−8 M level. Our SERRS sandwich-hybridization assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) for species detection with several application fields including: food frauds, forensics or studies of past populations through ancient DNA.


A novel SERRS sandwich-hybridization assay to detect specific DNA target.

Feuillie C, Merheb MM, Gillet B, Montagnac G, Daniel I, Hänni C - PLoS ONE (2011)

The sandwich-DNA hybridization assay principle.(A) Capture and detection probes are added to the solution of target DNA. (B) After the denaturation step (99°C, 10 minutes), the hybridization of the probes to the DNA target is achieved under gentle agitation (55°C, 3 hours). (C) Streptavidin-coated magnetic micro beads are added to the solution (gentle agitation for 30 minutes at room temperature). (D) Hybridized duplexes are captured by a magnet, unbound compounds are washed out. (E) Hybridized detection probes are eluted (95°C, 20 minutes) and are detected by SERRS in proportion of the initial concentration of target.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104981&req=5

pone-0017847-g001: The sandwich-DNA hybridization assay principle.(A) Capture and detection probes are added to the solution of target DNA. (B) After the denaturation step (99°C, 10 minutes), the hybridization of the probes to the DNA target is achieved under gentle agitation (55°C, 3 hours). (C) Streptavidin-coated magnetic micro beads are added to the solution (gentle agitation for 30 minutes at room temperature). (D) Hybridized duplexes are captured by a magnet, unbound compounds are washed out. (E) Hybridized detection probes are eluted (95°C, 20 minutes) and are detected by SERRS in proportion of the initial concentration of target.
Mentions: In this study, we have applied a Surface Enhanced Resonance Raman Scattering (SERRS) approach as an alternative technology to PCR amplification for the specific detection of DNA. SERRS is a vibrational spectroscopy technique whereby the Raman signal of the compound of interest can be amplified up to 1014 fold [23], [24]. SERRS-active molecules possess a chromophore with an absorption frequency close to the excitation frequency, and can adsorb on rough metallic surfaces such as colloidal silver nanoparticles. This adsorption has a doubly positive effect on the Raman signal: (i) it quenches the fluorescence that allows the highly specific Raman fingerprint of the molecule to be detected, (ii) it amplifies the Raman signal. Potential applications of SERRS detection have been under development since 1997 with a view of detecting DNA [25], thus becoming a rapidly emerging field [26]. Our present SERRS sandwich-hybridization assay is based on the specific hybridization of two nucleic probes to target DNA to be detected in solution (Figure 1). The nucleic probe labeled with rhodamine 6G (detection probe) allows the SERRS detection. The second probe, coupled with biotin (capture probe) allows immobilization and purification of the resulting hybridized complex (i.e. target DNA, capture and detection probes). Previous studies have demonstrated that SERRS-labeled synthetic DNA could be detected [25], [27], and that SERRS signal is stable after hybridization of a labeled oligonucleotide probe with a target DNA [26]. The sensitivity of SERRS makes it a valuable alternative non-enzymatic tool to detect DNA. We have developed a study model in which the target and control DNA are homologous sequences of chamois (Rupicapra rupicapra) and common goat (Capra hircus, a closely related species to R. rupicapra), respectively. The results of this new assay shows that DNA of R. rupicapra can be specifically discriminated from C. hircus at the 10−8 M level. Our SERRS sandwich-hybridization assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) for species detection with several application fields including: food frauds, forensics or studies of past populations through ancient DNA.

Bottom Line: In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA.In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors.As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Géologie de Lyon-Terre Planètes Environnement, ENS Lyon, Université Lyon 1, CNRS, Ecole Normale Supérieure de Lyon, Lyon, France.

ABSTRACT
In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics.

Show MeSH
Related in: MedlinePlus