ORIGINAL PAPER

Enzyme-free detection and quantification of double-strandednucleic acids

Cécile Feuillie & Maxime Mohamad Merheb &

Benjamin Gillet & Gilles Montagnac & Catherine Hänni &Isabelle Daniel

Received: 6 March 2012 /Revised: 24 April 2012 /Accepted: 21 May 2012 /Published online: 14 June 2012# Springer-Verlag 2012

Abstract We have developed a fully enzyme-free SERRShybridization assay for specific detection of double-strandedDNA sequences. Although all DNA detection methodsranging from PCR to high-throughput sequencing rely onenzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affectedby alterations, modifications, and/or quality of DNA. Forinstance, a limitation of most DNA polymerases is theirinability to process DNA damaged by blocking lesions. Asa result, enzymatic amplification and sequencing of degrad-ed DNA often fail. In this study we succeeded in detectingand quantifying, within a mixture, relative amounts of close-ly related double-stranded DNA sequences from Rupicaprarupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone

of a promising approach to overcome the failure of DNApolymerase when DNA is too degraded or when the con-centration of polymerase inhibitors is too high. It is the firsttime double-stranded DNA has been detected with a trulynon-enzymatic SERRS-based method. This non-enzymatic,inexpensive, rapid assay is therefore a breakthrough innucleic acid detection.

Keywords SERRS . Double-stranded DNA .

Non-enzymatic

Introduction

Genetic data extracted from a biological sample are usefulfor rapidly answering a wide range of questions rangingfrom ecology [1] to medicine, e.g., for monitoring viralRNA [2]. Genetic diagnostics has been constantly rein-vented, starting with Southern blotting in the 70s and simplePCR methods in the mid 80s [3, 4] to the last decade’s“next-generation” sequencing techniques. High-throughputsequencing methods [5, 6] and references cited therein pro-duce large high-quality data sets [5] enabling meta-genomeor genome-wide analysis of current and ancient samples [7,8]. Routinely, those methods rely on DNA polymerases foramplification of the target DNA before sequencing or forsequencing-by-synthesis. Unfortunately a major disadvan-tage is the inability of DNA polymerases to process some ofthe DNA alterations that occur in natural environments.After the death of an organism, and depending on preserva-tion conditions, DNA undergoes chemical modifications,for example strand breaks, oxidative damage, and molecularcrosslinks [9–13]. These post mortem modifications result

Cécile Feuillie and Maxime Mohamad Merheb contributed equally tothis work.

C. Feuillie (*) :G. Montagnac : I. DanielLaboratoire de Géologie de Lyon: Terre, Planètes, Environnement,UMR 5276, Université Lyon 1, CNRS,Ecole Normale Supérieure de Lyon,46 allée d’Italie,69364 Lyon Cedex 07, Francee-mail: cecile.feuillie@ens-lyon.org

M. M. Merheb :C. HänniInstitut de Génomique Fonctionnelle de Lyon, UMR 5242,Université Lyon 1, CNRS, Ecole Normale Supérieure de Lyon,46 allée d’Italie,69364 Lyon Cedex 07, France

B. GilletPlateforme nationale de Paléogénétique UMS PALGENE,CNRS - Ecole Normale Supérieure de Lyon,69366 Lyon Cedex 7, France

Anal Bioanal Chem (2012) 404:415–422DOI 10.1007/s00216-012-6133-1

from early digestion by endonuclease enzymes, and, later,from modifications resulting from environmental factors, forexample UV light, oxidation, and further action of biologi-cal scavengers [9–14]. Similar alterations also occur duringthe processing of food [15]. Among well-known types ofDNA damage [9], oxidized pyrimidines, abasic sites, andcross-links are called blocking lesions, because they stopthe DNA polymerase bypass and thus prevent amplifica-tion and/or sequencing [16–18]. New polymerases, forexample those of the Y-family or of the chimeric A-family that can bypass blocking lesions have recently beendeveloped [19, 20]. However, the engineering of damage-tolerant polymerases is a complex process and does notguarantee significantly higher amplification [18–20].Thus, a method for DNA detection truly independent ofenzymes would be of great value for rapid detection ofhighly degraded DNA.

We have developed a quick test to check for the presenceof a specific double-stranded DNA target in a sample. Thistest is fully non-enzymatic and relies on surface enhancedresonant Raman scattering (SERRS). SERRS is a vibration-al spectroscopic method that amplifies the Raman signal upto 1014 times [21, 22]. SERRS-labels are molecules withmaximum absorption close to the excitation wavelength andthat adsorb on rough metallic surfaces, for example silvercolloids [23] or nanostructured surfaces [24]. The fluores-cence of the SERRS-label is quenched by the metallicsurface enabling the amplified Raman fingerprint of themolecule to be observed. A wide range of labels can bedetected by SERRS, and the sharpness of Raman peaksgives SERRS high multiplexing capacity [25–30]. SERRShas been developed for DNA detection since 1997 [24, 31]and has become a promising enzyme-free tool for specificdetection and quantification of single-stranded DNA oneither micro-chips [30, 32, 33] or magnetic micro-beads[34, 35]. RNA can also be detected with a SERRS-baseddetection method for estimation of gene expression [33].Although several non-enzymatic methods exist for detectionof single-stranded nucleic acids, double-stranded nucleicacid detection is a greater challenge without use of enzymesbecause of the very high affinity of the target strand for itscomplementary strand. Hill et al. proposed, in 2007, a non-enzymatic assay based on hybridization with functionalizednanoparticles, silver enhancement, and scanometric detec-tion of double-stranded DNA [36]. Though highly efficientwith a detection limit of 2.5 fmol L−1, the latter assayrequires long multiple steps. Here we use SERRS technologyin a straightforward easy-to-use non-enzymatic procedure forrapid specific detection of double-stranded DNA. We alsotake advantage of the SERRS multiplexing capacity to suc-cessfully identify and quantify two closely related sequencesof double-stranded DNA in a mixture. This is the first time, tothe best of our knowledge, that double-stranded DNA has

been directly analyzed by a non-enzymatic SERRS-baseddetection method.

Results

Principle of specific DNA detection by SERRS

We set up a procedure for double hybridization and immo-bilization of target DNA on magnetic microbeads to detectdouble-stranded DNA (Fig. 1). We used two DNA targets: a92-base sequence of the mitochondrial DNA (12sRNAgene) of Rupicapra rupicapra (chamois) and the ortholo-gous sequence of 91 bases of Capra hircus (goat). Bothsequences were analyzed in a mixture with proportionsvarying from 0 % to 100 %. A biotinilated capture probe,that is a biotin-labeled oligonucleotide composed of 30bases, hybridizes with the target DNA at the 5′ end. Thecapture probe is identical for both R. rupicapra and C.hircus and enables immobilization of both target sequenceson streptavidin-coated magnetic microbeads. We used two26-base detection probes specific for each R. rupicapra andC. hircus target DNA and labeled with rhodamine 6G (R6G)and hexachlorocarboxyfluorescein (HEX), respectively.Both detection probes hybridize with their respective targetat the 3′ end. We added small blocking oligonucleotides inexcess both to prevent the rapid rehybridization of thecomplementary strands [36] and to enhance hybridizationof the capture and detection probes. We used two blockingoligonucleotides of 24 bases specific to R. rupicapra and C.hircus targets, respectively. After hybridization and immo-bilization on the magnetic microbeads, non-hybridized com-pounds are washed off. A final thermal dissociation stepenables recovery of the detection probes and their analysisby SERRS. This assay has the unique property of beingtotally non-enzymatic.

The multiplexing capacity of this assay lies in the avail-ability of a great variety of Raman labels, among which theHEX and R6G molecules were chosen. SERRS is a methodwith greater resolution than fluorescence, because Ramanpeaks are sharper. Consequently several SERRS-labeledoligonucleotides can be simultaneously detected if the ab-sorption maxima of the tags are close enough to the laserexcitation wavelength [25–30]. Maximum absorption ofHEX and R6G is at 535 and 524 nm, respectively, and cantherefore be simultaneously measured with an argon lasertuned at 514.5 nm [27, 29, 31, 37]. Moreover, theirRaman signals enable specific discrimination of eachprobe, because of its most intense peaks centered at1632 cm−1 and 1650 cm−1 for HEX and R6G, respec-tively (Fig. 2). Those peaks correspond to the combinedsignature of xanthene ring stretching and in plane C–Hbending modes [38].

416 C. Feuillie et al.

Specific detection and quantification of double-strandedDNA

We investigated mixtures of double-stranded DNA sequen-ces from mitochondrial DNA (12sRNA) of Rupicapra rupi-capra (chamois, 92 bp) and Capra hircus (common goat,

91 bp) by the procedure depicted in Fig. 1. We achievedspecific detection of both double-stranded DNA targets insolution with final DNA target concentrations between 5×10−9 and 10−7 mol L−1 and variable relative quantities of R.rupicapra and C. hircus. The resulting SERRS spectra for afinal DNA target concentration of 5×10−8 mol L−1 aredisplayed in Fig. 2. When the sample contains 100 %double-stranded goat DNA (C. hircus), the spectrum con-tains only the fingerprint of the HEX probe with the mostintense peak at 1632 cm−1 (Fig. 2). This indicates that theR6G probe, characterized by an intense peak at 1650 cm−1,does not hybridize with C. hircus DNA. In contrast, thespectrum of a sample that contains 100 % of chamoisdouble-stranded DNA contains only the Raman signal ofR6G characterized by the intense peak at 1650 cm−1

(Fig. 2). No signal is observed at 1632 cm−1, indicating thatthe HEX probe does not hybridize with R. rupicapra. Whenboth DNA targets are mixed spectra contain signals fromboth R6G and HEX-labeled probes (Fig. 2). The more R.rupicapra target DNA, the more intense the peak centered at1650 cm−1. Similarly, the more C. hircus target DNA, themore intense the peak centered at 1632 cm−1. The respectivesignal intensity of each probe is positively correlated withthe amount of specific DNA target. In the absence of targetDNA the SERRS spectrum of the control is void of R6G andHEX signal indicating that the SERRS probes have beenfully removed during the washing step (Fig. 2). More impor-tantly, it confirms the specificity of this SERRS hybridization

Fig. 1 SERRS assay developed in this study. Capture and detectionprobes and blocking oligonucleotides are added to a sample of double-stranded target DNA. After denaturation (95 °C, 2 min), rehybridiza-tion of target DNA is prevented by the presence of small oligonucleo-tides added in excess (103-fold), and the probes hybridize to the targets(55 °C to 25 °C, 1 degree per min). The hybridized complexes areimmobilized on streptavidin-coated magnetic micro beads, and

unbound compounds are washed off. Hybridized detection probes areeluted by thermal dissociation (95 °C, 20 min) and are detected bySERRS in proportion to the initial concentrations of the targets. In thisstudy, target 1 was R. rupicapra and target 2 was C. hircus. Theorange-labeled detection probe is the R6G probe specific to R. rupi-capra and the pink-labeled detection probe is the HEX probe specificto C. hircus. These assays are suitable for RNA detection also

Fig. 2 Specific SERRS signals obtained. Mixtures of two double-stranded DNA targets, from R. rupicapra and C. hircus, were analyzedby the SERRS assay developed in this study. The final target concen-tration was 5×10−8 mol L−1 and the relative abundance of each targetvaried between 0 and 100 %. The peak at 1632 cm−1, characteristic ofthe HEX-detection probe, shows the presence of C. hircus. The peak at1650 cm−1, characteristics of R6G, shows the presence of R. rupicapra.The n − 100−n labels on the right indicate the relative amount of C.hircus and R. rupicapra, respectively

Enzyme-free detection and quantification of ds DNA 417

assay, because we do not observe any non-specific hybridiza-tion or adsorption. The limit of detection (LOD) was calcu-lated from the spectra acquired with a final targetconcentration of 5×10−8 M, in accordance with Haller andKnochel in 1996 [39]. LODs are 3.2×10−10 mol L−1 and 4.5×10−10 mol L−1 for R. rupicapra and C. hircus, respectively.

For quantification we consider the area of the most in-tense peak characteristic of each labeled probe, at 1650 cm−1

for the R6G probe and at 1632 cm−1 for the HEX probe,hereafter noted A1650 and A1632, respectively [24, 35]. Weuse the Peakfit software to process the spectra. Fits of thepeaks are displayed in Fig. 3a, below the raw spectrum.The peak areas quantify the relative amount of eachprobe R6G/(R6G+HEX) in the form of the ratio A1650/(A1650+A1632) measured in the spectrum. Figure 4 is aplot of the percentage of R. rupicapra DNA in theanalyzed mixture as a function of the ratio A1650/(A1650+A1632). It is well described by the relationship:

%Rup ¼ 95:77 �5:78ð Þ A1650= A1650 þ A1632ð Þ½ �2;R2 ¼ 0:94

Residuals are also displayed in Fig. 4, and indicate thatthe SERRS assay developed in this study enables identifi-cation of the DNA components of a mixture with uncertain-ty better than 15 %.

The challenge in analyzing double-stranded DNA by useof a hybridization assay is to avoid the very rapid rehybrid-ization of the target strand with its complementary strand. Inthis study this problem was circumvented by the use ofsmall oligonucleotides as blockers to inhibit the rehybrid-ization of the double-stranded DNA target. Because of theirsmall size compared with the target and their high

Fig. 3 Effect of the use of blocking oligonucleotides. (a) In black, thenegative control with no target DNA. In blue, a mixture of 75 % R.rupicapra, 25 %C. hircus, without blockers. In red, the same mixture,with blockers. The final target concentration is 5×10−8 mol L−1. Fittedpeaks are depicted below the spectrum. (b) Intensity of the SERRSsignal with (red) or without (blue) blocking oligonucleotides for themixture of 75 % R. rupicapra and 25 % C. hircus studied in Fig. 3a.Error bars correspond to the standard deviation

Fig. 4 Quantification of the relative amount of target DNA. Percent-age of R. rupicapra DNA in the analyzed mixture as a function of the

ratio A1650 ;R6G

A1650; R6GþA1632 ;HEXð Þ measured in the SERRS spectrum. Relative

abundance is determined with 15 % uncertainty

418 C. Feuillie et al.

concentration, the oligonucleotides hybridize much faster tothe target DNA than the complementary strand, leaving thetarget strand available for hybridization with the probes.This method was first proposed by Hill et al. in 2007, whoworked on genomic DNA from B. subtilis 168 using threeblockers at a concentration 106 times in excess. We im-proved this procedure by using only one blocker instead ofthree, and by minimizing the blocker’s concentration bythree orders of magnitude. The blockers were added at aconcentration 103 times in excess to the total target DNA.We checked that the blockers were not a source of non-specific hybridization or adsorption. Solutions containingthe capture probe, the detection probes, and both blockerswere analyzed by the procedure depicted in Fig. 1. Thecorresponding spectrum is displayed in Fig. 3a (black spec-trum) and shows neither the signal of R6G nor that of HEX.This indicates that blocking oligonucleotides do not non-specifically hybridize with the detection probes and willtherefore not lead to false-positive results. To emphasizethe importance of blockers in this assay, we analyzeddouble-stranded DNA mixtures with a final concentrationof 5×10−8 mol L−1 with and without blockers. SERRSspectra obtained for mixtures containing 25 % R. rupicapraand 75 % C. hircus DNA are displayed in Fig. 3a. In asolution void of blockers but completed with thecorresponding volume of buffer we observe weak signalsfrom both detection probes (Fig. 3a, blue spectrum). Whenblockers are added 103 times in excess, the intensity of thesignals increases strikingly (Fig. 3b, red spectrum). Meanareas of the Raman peaks of the R6G probe and of the HEXprobe specific to R. rupicapra and C. hircus, respectively,are presented in Fig. 3b. The area of the R6G-characteristicpeak centered at 1650 cm−1 is increased by 300 % and thearea of the HEX-characteristic peak centered at 1632 cm−1

increases by 230 %. The use of blocking oligonucleotidestherefore furnishes a SERRS signal that is high enough inintensity to be processed and interpreted. We have suc-ceeded in detecting and quantifying specific double-stranded DNA sequences without the need for an enzymaticstep. As enzymatic trials may be tedious and inconclusive,this rapid and inexpensive assay is a quick means of obtain-ing valuable spectroscopic information and could save timeand money.

Conclusion

We have developed a truly enzyme-free SERRS assay, com-bined with use of blockers, that enables specific detectionand quantification of double-stranded DNA. We succeededin identifying DNA sequences from two closely relatedspecies in a mixture. The measured SERRS spectra can beused to quantify the relative amounts of each DNA target

with only 15 % uncertainty. Being totally non-enzymaticthis assay has the potential to challenge PCR or polymerase-based DNA detection techniques. Identification of specificDNA molecules would be possible in one trial even whenextracted DNA is degraded and, therefore, most of the timeunavailable for amplification or sequencing. This rapid andsimple assay enables processing of samples in ca 2 h only,and is very complementary to the highly sensitive but morecomplex method of Hill et al. [36]. Moreover, our methodprovides a way to detect short DNA fragments under 100base pairs by a fully non-enzymatic approach. This SERRSassay is highly valuable for checking for the presence ofspecific DNA, and concentrating it prior to more thoroughinvestigation. Finally, as this assay relies on hybridization, itwould not be sensitive to DNA polymerase inhibitors andcould be used on a wider range of samples without time-consuming purification steps, often critical for retrieval ofDNA.

Many fields could take advantage of this assay, in partic-ular forensics [40], food fraud detection [15], ecology [41],and paleogenetics [42–44], and, more generally, wheneverDNA is fragmented, degraded, and co-extracted with poly-merase inhibitors. Moreover, this SERRS DNA detectionassay could also become a versatile tool for medical diag-nosis, for instance to monitor RNA expression by tumorcells. In a near future, this non-enzymatic double-strandedDNA detection method could be applied to a whole newrange of samples containing refractory DNA.

Materials and methods

DNA sequences and oligonucleotide probes

We used two DNA targets: a 92 bases sequence of mito-chondrial DNA (12sRNA gene) of Rupicapra rupicapra(chamois) and the homologous sequence of 91 bases ofCapra hircus (goat). Chamois and goat were chosen becausethey are closely related and because neither is a usualsample contaminant (e.g. pig Sus scrofa, bovid Bos taurus,chicken Gallus gallus, …) [45].

Target DNA sequences, the SERRS 26-mer oligonucleo-tide labeled with either one molecule of rhodamine 6G(R6G) or one molecule of hexachlorocarboxyfluorescein(HEX) (detection probe), the biotin labeled 30-mer oligonu-cleotide (capture probe), and the blocker oligonucleotides,small oligonucleotides of 24 bases, were purchased fromEurogentec. Sequences were aligned using Seaview [46].The 30-mer biotin probe was modified with 3′-biotin, whichwas linked to the oligonucleotide via the nine-atom spacertriethylene glycol (TEG) which provides the biotin withgood accessibility to the streptavidin-coated magneticmicrobeads. The 26-mer R6G-probe was modified with

Enzyme-free detection and quantification of ds DNA 419

5′-Rhodamine 6G (R6G), which is linked to the oligo-nucleotide via a C6 spacer. The HEX-probe is modifiedwith 5′-HEX, the hexachloro derivative of carboxyfluor-escein, also linked to the oligonucleotide via a C6spacer.

The principle of the assay is summarized in Fig. 1.All DNA sequences used in this study are summarized in

Table 1.

SERRS-labels

Several SERRS-labeled oligonucleotides can be simulta-neously detected [25–30]. A variety of molecules can bedetected by SERRS if their absorption maxima are closeenough to the laser excitation wavelength. We choseR6G and HEX as SERRS-labels in our assay for threereasons. HEX and R6G have absorption maxima at 535and 524 nm, respectively, and can therefore be simulta-neously detected by use of an argon laser tuned at514.5 nm [27, 29, 31, 37]. Moreover, their detectionlimits are close, 1.2×10−12 mol L−1 and 7.8×10−12 mol L−1

for R6G and HEX, respectively [27, 29, 31]. Finallytheir Raman signals enable specific discrimination ofeach probe because of their most intense peaks centeredat 1632 cm−1 and 1650 cm−1 for HEX and R6G, re-spectively (Fig. 2). Those peaks correspond to the com-bined signature of xanthene ring stretching and in planeC–H bending modes [38].

Reagents

All reagents were analytical grade. Tetrahydrochloride sper-mine (Fluka, #85610), polyoxyethylenesorbitan monolau-rate (Tween 20, #P1379), and silver nitrate 99.999 %(#S8157) were purchased from Sigma–Aldrich. Trisodiumcitrate (1 %; #S1804) was from Fisher. Ultra pure 20×SSCBuffer (Gibco, #15557-044), streptavidin-coated magnet-ic microbeads (Dynal, Dynabeads MyOne StreptavidinC1, #650-02, 10 mg mL−1, 2 mL, 7–12×109 beads), and thematching DynaMag-2 magnetic separator (Dynal, #123-21D)were purchased from Invitrogen.

Preparation of double-stranded target DNA

Complementary strands of both DNA targets were specifi-cally hybridized before the experiment. Solutions (50 μL) ofeach complementary strand at a concentration of 2×10−7 mol L−1 were mixed, and left to stand at room temper-ature for at least 60 h, furnishing a solution of double-stranded DNA at a concentration of 10−7 mol L−1. Concen-tration was adjusted in accordance with to the manufac-turer’s instructions.

Hybridization of target with capture and detection probes

Hybridization was performed in a single step as describedhereafter for mixtures of both target DNA in different

Table 1 DNA sequences used in this study. Mismatches between the targets are displayed in red. Boxes represent the hybridization zones for (1)the capture probe, (2) the R6G-detection probe, (3) the HEX detection probe, (4) the R. rupicapra blocker, and (5) the C. hircus blocker

420 C. Feuillie et al.

relative amounts. Target DNA solution (10 μL; concentra-tion ranging from 5×10−9 to 10−7 mol L−1) were mixed with10 μL of the capture probe, 10 μL of each detection probe(both in excess×10, probe solution concentrations depend-ing on the concentration of target DNA), and 10 μL of eachblocking oligonucleotide. Final experimental volume was60 μL. The blocking oligonucleotides were initially pre-pared in 4×SSC, Tween 20 (0.5 %). Then, the resultingsolution was heated to 95 °C for 2 min to ensure denatur-ation of the double-stranded DNA target, potential hairpins,or autohybridized DNA. Hybridization was achieved in athermocycler, by lowering the temperature from 55 °C to25 °C at a rate of 1 ° min−1.

Immobilization

Meanwhile, series of 25 μL of the stock solution(10 mg mL−1) of magnetic microbeads were rinsed threetimes in 25 μL (4×SSC, 0.5 % Tween 20) buffer using themagnetic particle concentrator (MPC). Microbeads werethen resuspended in 10 μL buffer and added to the hybrid-ization solution for immobilization, with gentle continuousstirring for 30 min at room temperature. The beads werefinally washed twice in 150 μL 0.1×SSC, Tween 20 (0.5 %)buffer using the MPC to remove the unbound material.Several washing buffer salinities were tested ranging from0.05×SSC to 4×SSC, and a salinity of 0.1×SSC was cho-sen to optimize the specificity of our assay without anysignificant loss of material.

Beads re-suspended in 60 μL 0.1×SSC, 0.5 % Tween20 buffer were finally heated at 95 °C for 20 min fordenaturation of both the DNA sandwich and the biotin-streptavidin bound. The microbeads were then immobi-lized by the MPC and the supernatant was collected forSERRS measurements.

SERRS measurements

The silver colloid used for SERRS measurements was pre-pared in accordance with the Lee and Meisel procedure [47].Silver nitrate (90 mg) was dissolved in 500 mL distilledwater and subsequently heated, with continuous stirring,until boiling. Sodium citrate solution (1 %, 10 mL) was thenadded. The solution was maintained at boiling for 90 min.The silver colloid had been stored in the dark at roomtemperature and the silver colloid aliquots used in this studywere from the same batch. The eluted R6G probes containedin the supernatant were analyzed by following the procedureof Feuillie et al. [35]. The supernatant (20 μL) was mixedwith 20 μL spermine (10−2 mol L−1) in a single-use PMMAspectroscopy cuvette. The spermine was used as an aggre-gating agent to reduce the electrostatic repulsion betweenthe silver nanoparticles. Silver colloid (500 μL) and distilled

water (500 μL) were then added and the solution washomogenized before SERRS measurement.

Samples were analyzed with a visible Horiba Jobin YvonLabRam HR 800 Raman spectrometer, coupled to a SpectraPhysics 2018 Ar+/Kr+ 24 laser tuned at 514.5 nm (LGL,ENS de Lyon). The laser power used on the sample wasselected in a range between 1.5 and 2 mW. The spectra wereacquired with a spectrometer grating of 1800 gr mm−1 cen-tered at 1600 cm−1. Spectra were the result of 1×10 to 1×60 s accumulation, depending on the concentration of theSERRS probe.

Quantification of the relative amount of both DNA targets

The variable chosen for quantification was the area of themost intense peak characteristic of each labeled probe, at1650 cm−1 for the R6G probe and at 1632 cm−1 for the HEXprobe, hereafter denoted A1650 and A1632, respectively [24,35].

SERRS spectra were processed with Peakfit software.The fits obtained for the peaks are displayed in Fig. 3abelow the raw spectrum. The peak areas enable quantifica-tion of the relative amounts of each probe R6G/(R6G +HEX) in the form of the ratio A1650/(A1650+A1632) measuredin the spectrum. This ratio is positively correlated with therelative amount of R. rupicapra target in the initial solution.

Acknowledgements The authors are grateful to the Paleogenomicsand Molecular Evolution team at IGFL for technical support and helpfulcomments. We warmly thank S. Hughes and J. Burden for improving themanuscript. The project was supported by the Interdisciplinary Programof CNRS “Interface physique, biologie et chimie: soutien à la prise derisque”, the Interdisciplinary program of Ecole Normale Supérieure deLyon and by the Région Rhone-Alpes CIBLE 2011.

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