Probes and DNA characteristics.

<p>(A) Probes and DNA sequences. (B) Alignment of Non target DNA (<i>C. hircus</i>) sequence with complement sequences of Capture probe and Detection probe. Numbers present non-effective bases in the Non target DNA sequence.</p

SERRS-hybridization detection results.

<p>a) SERRS spectra obtained for the analysis of the non-degraded double stranded molecule N_5′/N_3′ (in red), and a control solution void of target DNA (in black). The parameter used for quantification is the area of the most intense Raman band of Rhodamine 6G at 1650 cm⁻¹, and is noted A₁₆₅₀. Target: 5×10⁻⁸M; - Blockers: 5×10⁻⁵M; - Capture and detection probe: 10 mM. The peaks visible on the control spectrum come from the PMMA cuvettes used for the measurements. b) SERRS-hybridization assay detection results. In grey is the SERRS signal obtained for the single-stranded non-degraded DNA sequence N_5′. In red is the SERRS signal obtained for the detection of the non-degraded double-stranded N/N molecule. Degraded molecules appear in orange, except for molecules containing the V_5′ strand with 5 abasic sites, that appear in blue. All degraded molecules are detected with signals comparable to those of non-degraded molecules. Concentrations used in this study: - Target: 5×10⁻⁸M; - Blockers: 5×10⁻⁵M; - Capture and detection probe: 10 mM. Error bars are 2 standard deviation.</p

The sandwich-DNA hybridization assay principle.

<p>(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.</p

Combination of double-stranded R_5′/R_3′ molecules investigated in this study.

<p>N_5′/N_3′ is the original non-degraded molecule.</p><p>Combination of double-stranded R_5′/R_3′ molecules investigated in this study.</p

Specificity and co-contamination assays.

<p>All spectra were acquired in 1 acquisition of 30 s. (A) In black, negative control without target DNA. (B) In blue, <i>C. hircus</i>, negative control (5.10⁻⁸ M). (C) In green, signal from <i>R. rupicapra</i> (5.10⁻⁸ M). (D) In red, signal obtained from an equimolar mix of <i>R. rupicapra</i> and <i>C. hircus</i> DNA (5.10⁻⁸ M each).</p

Nucleic sequences used in this study.

<p>5′ and 3′ subscripts indicate sequences in the 5′-3′ and 3′-5′ orientations, respectively. Sequences are aligned, and abasic sites (AS) are labeled in red with their positions. PCR primers Rup_For_5′ and Rup_Rev_3′ and their alignment to target sequences are also represented. SERRS capture probe, detection probe and 3 blockers are labeled Cap_3′, Det_3′, Block1_5′, Block2_5′ and Block3_5′, respectively.</p><p>Nucleic sequences used in this study.</p

SERRS-hybridization assay principle of double-stranded DNA detection.

<p>One strand is specifically hybridized to 2 probes, a biotin-labeled capture probe and a R6G-labeled detection probe. The second strand is specifically hybridized to three oligonucleotides which block the rehybridization to its complementary strand.</p

High-resolution agarose gel electrophoresis of PCR products obtained from amplification of eleven double-stranded DNA molecules containing abasic sites.

<p>PCR conditions are given in the “<i>Material and Methods: PCR conditions</i>” part. Four independent PCR reactions were carried out for each degraded DNA template using Rup_For_5′ and Rup_Rev_3′ primers (expected fragment size  =  134 bp). The size of some DNA markers in bp is indicated. All gels were revealed using the same transilluminator settings.</p