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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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Related in: MedlinePlus

Probes and DNA characteristics.(A) Probes and DNA sequences. (B) Alignment of Non target DNA (C. hircus) sequence with complement sequences of Capture probe and Detection probe. Numbers present non-effective bases in the Non target DNA sequence.
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pone-0017847-g004: Probes and DNA characteristics.(A) Probes and DNA sequences. (B) Alignment of Non target DNA (C. hircus) sequence with complement sequences of Capture probe and Detection probe. Numbers present non-effective bases in the Non target DNA sequence.

Mentions: To assess the specificity of the sandwich-hybridization assay presented in this study, we also tested the homologous region of mitochondrial DNA (12sRNA gene, 138 bp) of the domestic goat (Capra hircus) against chamois (R. rupicapra, 12sRNA gene, 139 bp) for which the SERRS detection was positive as presented above. More precisely, the sequence of C. hircus displays 4 mismatches with the capture probe, and 3 mismatches with the detection probe (Figure 4B). Samples containing either DNA of C. hircus only, or an equimolar mix of R. rupicapra and C. hircus DNA were investigated. The SERRS spectra obtained with 5×10−8 M C. hircus samples are similar to those of the negative control obtained without target DNA (Figure 3B). The absence of Raman signal from R6G clearly indicates that SERRS probes have been fully washed out during the elution process and confirms the specificity of the present sandwich hybridization assay (no unspecific/a-specific hybridization). Moreover, the Raman spectrum of the sample containing both R. rupicapra and C. hircus displays the same signal as the R. rupicapra sample only (Figure 3D and 3C respectively). This indicates that when the DNA (C. hircus in this study) present in a sample is not specific to the capture and detection probes (specific to R. rupicapra in this study), or when no DNA is present, the R6G SERRS signal is not observed in the elution solution. On the contrary, when the solution contains target DNA, with or without non-target DNA, a clear SERRS signal is observed (Figure 3), that allows quantification of the amount of target 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)

Probes and DNA characteristics.(A) Probes and DNA sequences. (B) Alignment of Non target DNA (C. hircus) sequence with complement sequences of Capture probe and Detection probe. Numbers present non-effective bases in the Non target DNA sequence.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017847-g004: Probes and DNA characteristics.(A) Probes and DNA sequences. (B) Alignment of Non target DNA (C. hircus) sequence with complement sequences of Capture probe and Detection probe. Numbers present non-effective bases in the Non target DNA sequence.
Mentions: To assess the specificity of the sandwich-hybridization assay presented in this study, we also tested the homologous region of mitochondrial DNA (12sRNA gene, 138 bp) of the domestic goat (Capra hircus) against chamois (R. rupicapra, 12sRNA gene, 139 bp) for which the SERRS detection was positive as presented above. More precisely, the sequence of C. hircus displays 4 mismatches with the capture probe, and 3 mismatches with the detection probe (Figure 4B). Samples containing either DNA of C. hircus only, or an equimolar mix of R. rupicapra and C. hircus DNA were investigated. The SERRS spectra obtained with 5×10−8 M C. hircus samples are similar to those of the negative control obtained without target DNA (Figure 3B). The absence of Raman signal from R6G clearly indicates that SERRS probes have been fully washed out during the elution process and confirms the specificity of the present sandwich hybridization assay (no unspecific/a-specific hybridization). Moreover, the Raman spectrum of the sample containing both R. rupicapra and C. hircus displays the same signal as the R. rupicapra sample only (Figure 3D and 3C respectively). This indicates that when the DNA (C. hircus in this study) present in a sample is not specific to the capture and detection probes (specific to R. rupicapra in this study), or when no DNA is present, the R6G SERRS signal is not observed in the elution solution. On the contrary, when the solution contains target DNA, with or without non-target DNA, a clear SERRS signal is observed (Figure 3), that allows quantification of the amount of target 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