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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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Specific SERRS signals.(A) SERRS spectra of the probe labeled with a single rhodamine 6G (R6G). The peak area beneath the most intense peak at 1650 cm−1 (A1650) is the parameter chosen for quantification. The black spectrum is the Raman signal of the PMMA cuvette, appearing in all spectra. Acquisition times for 0 M, 5×10−9 M, 5×10−8 M and 5×10−7 M are 1×15 s, 1×30 s, 1×10 s and 1×10 s, respectively. (B) Dilution study achieved on solutions of R6G probe. Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of the R6G probe (CR6G). Gray line represent the 1σ deviation. (C) Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of target DNA Gray lines represent the 1σ deviation.
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pone-0017847-g002: Specific SERRS signals.(A) SERRS spectra of the probe labeled with a single rhodamine 6G (R6G). The peak area beneath the most intense peak at 1650 cm−1 (A1650) is the parameter chosen for quantification. The black spectrum is the Raman signal of the PMMA cuvette, appearing in all spectra. Acquisition times for 0 M, 5×10−9 M, 5×10−8 M and 5×10−7 M are 1×15 s, 1×30 s, 1×10 s and 1×10 s, respectively. (B) Dilution study achieved on solutions of R6G probe. Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of the R6G probe (CR6G). Gray line represent the 1σ deviation. (C) Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of target DNA Gray lines represent the 1σ deviation.

Mentions: A concentration study of the detection probe (i.e. nucleic probe labeled with the R6G dye) was performed for concentrations ranging from 10−10 to 10−6 M, without target DNA. The Raman fingerprint of the R6G probe and of the polymethyl methacrylate (PMMA) cuvette are displayed in Figure 2A. R6G is characterized by 9 Raman peaks, centered at 640, 774, 1130, 1185, 1312, 1363, 1510, 1571 and 1650 cm−1, respectively. Figure 2B plots the peak area beneath the most intense Raman band at 1650 cm−1, hereafter noted A1650, normalized to acquisition time t, as a function of the CR6G concentration of the detection probe. The ratio A1650/t varies linearly with the CR6G concentration, with a slope of 2.13(9)×109 cts.s−1.M−1. This shows that the peak area beneath the most intense Raman band at 1650 cm−1 (A1650) allows quantification of the amount of DNA in the solution of interest.


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)

Specific SERRS signals.(A) SERRS spectra of the probe labeled with a single rhodamine 6G (R6G). The peak area beneath the most intense peak at 1650 cm−1 (A1650) is the parameter chosen for quantification. The black spectrum is the Raman signal of the PMMA cuvette, appearing in all spectra. Acquisition times for 0 M, 5×10−9 M, 5×10−8 M and 5×10−7 M are 1×15 s, 1×30 s, 1×10 s and 1×10 s, respectively. (B) Dilution study achieved on solutions of R6G probe. Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of the R6G probe (CR6G). Gray line represent the 1σ deviation. (C) Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of target DNA Gray lines represent the 1σ deviation.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104981&req=5

pone-0017847-g002: Specific SERRS signals.(A) SERRS spectra of the probe labeled with a single rhodamine 6G (R6G). The peak area beneath the most intense peak at 1650 cm−1 (A1650) is the parameter chosen for quantification. The black spectrum is the Raman signal of the PMMA cuvette, appearing in all spectra. Acquisition times for 0 M, 5×10−9 M, 5×10−8 M and 5×10−7 M are 1×15 s, 1×30 s, 1×10 s and 1×10 s, respectively. (B) Dilution study achieved on solutions of R6G probe. Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of the R6G probe (CR6G). Gray line represent the 1σ deviation. (C) Calibration curve of A1650 normalized to the acquisition time in second, as a function of the initial concentration of target DNA Gray lines represent the 1σ deviation.
Mentions: A concentration study of the detection probe (i.e. nucleic probe labeled with the R6G dye) was performed for concentrations ranging from 10−10 to 10−6 M, without target DNA. The Raman fingerprint of the R6G probe and of the polymethyl methacrylate (PMMA) cuvette are displayed in Figure 2A. R6G is characterized by 9 Raman peaks, centered at 640, 774, 1130, 1185, 1312, 1363, 1510, 1571 and 1650 cm−1, respectively. Figure 2B plots the peak area beneath the most intense Raman band at 1650 cm−1, hereafter noted A1650, normalized to acquisition time t, as a function of the CR6G concentration of the detection probe. The ratio A1650/t varies linearly with the CR6G concentration, with a slope of 2.13(9)×109 cts.s−1.M−1. This shows that the peak area beneath the most intense Raman band at 1650 cm−1 (A1650) allows quantification of the amount of DNA in the solution of interest.

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