Generic Platform for the Multiplexed Targeted Electrochemical Detection of Osteoporosis-Associated Single Nucleotide Polymorphisms Using Recombinase Polymerase Solid-Phase Primer Elongation and Ferrocene-Modified Nucleoside Triphosphates

Osteoporosis is a multifactorial disease influenced by genetic and environmental factors, which contributes to an increased risk of bone fracture, but early diagnosis of this disease cannot be achieved using current techniques. We describe a generic platform for the targeted electrochemical genotyping of SNPs identified by genome-wide association studies to be associated with a genetic predisposition to osteoporosis. The platform exploits isothermal solid-phase primer elongation with ferrocene-labeled nucleoside triphosphates. Thiolated reverse primers designed for each SNP were immobilized on individual gold electrodes of an array. These primers are designed to hybridize to the SNP site at their 3′OH terminal, and primer elongation occurs only where there is 100% complementarity, facilitating the identification and heterozygosity of each SNP under interrogation. The platform was applied to real blood samples, which were thermally lysed and directly used without the need for DNA extraction or purification. The results were validated using Taqman SNP genotyping assays and Sanger sequencing. The assay is complete in just 15 min with a total cost of 0.3€ per electrode. The platform is completely generic and has immense potential for deployment at the point of need in an automated device for targeted SNP genotyping with the only required end-user intervention being sample addition.


Tables
. Sequences of the primer sets used in the final assay and the sequences containing the SNP site. (There are two SNP-related specific primers and two other primers (with non-SNP specific terminal bases) used as negative controls.) 11 Table S2. TaqMan probes and the SNPs to be detected. 14 Table S3. Designed DNA sequences and primers for Sanger sequencing (primers are underlined and the SNP under interrogation is italicised).  (Table S1). β-globin was used as positive control of the reaction. 25 Figure S6. A) Mapping of electrode array provided by LABMAN and the pattern of electrode array functionalisation. The SNP related primers are highlighted in different colours (red for primers ending in T for SNPs A, blue for primers ending in C for SNPs G and green for primers ending in G for SNPs C) while the negative control primers are represented in grey and black colours). Finally, the positive controls (β-globin and Poly-Fc) are in pink and magenta colours. B) Real picture of electrode array during functionalisation process containing the drops over the electrodes. 26  Table S1. 28 Figure S10. SWV recorded in 0.1 M Sr(NO 3 ) 2 + 0.1 M Glycine pH 3 for simultaneous electrochemical detection of 5 SNPs for 10 more human whole blood samples. The SNP related primers are highlighted in different colours (red for primers ended in T for SNPs A, blue for primers ended in C for SNPs G and green for primers ended in G for SNPs C) while the negative primers are represented black colour and discontinuous traces. 38 Figure S11. TaqMan fluorogenic 5-exonuclease assay for SNP 10, SNP 27, SNP 29, SNP 46 and SNP 49: A) Allelic discrimination plots show three clusters with assigned genotypes as well as a zone for the no template controls (NTCs), shown in red, near the origin. Fluorescent endpoint data points in each cluster are grouped closely together and each cluster is located well away from the other clusters as well as from the NTCs. Data Figure S12. Chromatograms of the SNPs region obtained by Sanger sequencing using the Bioedit software. 44 Figure S13. Alignment of the obtained sequences by Sanger sequencing compared with their synthetic sequence DNA using Blastn software. 74 Figure S14: Plot representing data detailed in Table 3. Cases 9, 10 represent cases with a high risk of developing osteoporosis.

Materials
The chemical reagents were used as received. and imaged with a UV lamp (λ = 254 nm).

Primer design and evaluation
The sequences of the corresponding SNPs were found using the SNPedia website Blast was used to obtain primers with similar Tm, GC content and to produce amplicons ranging from 80 to 150 bp (Table S1). The specificity of the designed primers was confirmed using the "nr" parameter ( Figure S2). The Multiple Primer Analyzer software, was used to ensure that no self-dimers or primer-dimers would be obtained ( Figure S3). Briefly, 50 μL of RPA reagents (1 × rehydration buffer, 1 × basic E-mix, 1 × core reaction mix, 0.5 μM of each forward primer, 0.5 μM of the desired reverse primer, 0.2 mM dNTPs, 10 mM Mg(OAc) 2 ), 100 pM of dsDNA of the desired target (dsDNA prepared by PCR as described in the next section) were mixed and incubated for 15 min at 37 °C.
Prior to running the samples in a 2.6% agarose gel electrophoresis the RPA reaction was stopped by heating the samples to 80 °C for 10 min.

Double stranded DNA targets from synthetic sequences for optimisation studies
For optimisation of the RPA parameters, double stranded DNA sequences produced using PCR were used to mimic the amplicons that would be obtained from the genomic DNA target. 4 Using the PCR protocol described above, five individual PCR reactions were performed using the specific pair of forward and reverse primers for each synthetic amplicon (Table S1). The products were purified using the DNA Clean and Concentrator kit, visualised by gel electrophoresis and quantified by SimpliNano spectrophotomer. The generated double stranded DNAs were stored at -20°C until use.

Real samples used for genomic sensor validation
Fifteen human whole blood samples were selected from the Biobank of the Medical University of Graz, Austria. Genomic DNA was extracted and purified from five of these samples following NucleoSpin Blood Kit (Machery and Nagel 740951.250) to evaluate the effect of the blood matrix on the isothermal solid-phase primer elongation approach used for SNP detection.

Solid phase RPA for simultaneous detection of 5 SNP related to osteoporosis on maleimide activated plate and colorimetric detection
Firstly, the maleimide activated plate was washed three times with PBS-Tween. The five sets of thiolated reverse primers (Table S1:

Solid phase amplification based on electrode assay
The electrode arrays were designed at URV using AUTOCAD software ( Figure S7). Samtec connector is soldered to a carrier PCB to enable wired connections. Inside the break-out enclosure, wired connections were made from the Samtec connector PCB to individual standard 4 mm sockets. As well as WE, CE and RE connections and alignment test points, a chassis ground was produced to reduce extraneous electrical noise.
All the electrochemical measurements were carried out using an Autolab model PGSTAT 12 potentiostat/galvanostat controlled by the General Purpose Electrochemical System (GPES) software (Eco Chemie B.V., the Netherlands) and a Multiplexer module for 64 electrodes.

Electrode array functionalisation
The electrode array was washed with 50 % v/v isopropanol in water, followed by washing with MilliQ water and drying with nitrogen. The electrode functionalisation was carried out following the pattern outlined in Figure S6A and using the primers listed in Table S1.
The functionalisation of individual electrodes of the array was carried out by drop-casting 0.5 µL of 5 µM primer with 50 µM 6-mercaptohexanol in 1 M KH 2 PO 4 solution, and incubating for 3 h at 37 °C inside a humidity chamber ( Figure S6B). Finally, the electrode array was washed with water, dried under nitrogen and stored at 4 °C until use.

Electrochemical detection of solid-phase primer elongation
As previously explained, three types of targets were used in different steps of this study, amplicons generated from synthetic DNA targets (Table S1)

SNP detection using TaqMan fluorogenic 5-exonuclease assay.
TaqMan SNP genotyping assays were used as a reference method. In order to achieve optimal discrimination results, PCR reaction mixes were pre-tested with two mastermixes  (Table S2). Endpoint fluorescence was measured in a Polarstar Optima plate reader (BMG Labtech, Ortenberg, Germany).
Fluorescent data were exported into excel format and analyzed as scatter plots ( Figure   S11).

Sanger sequencing
PCR products were required to carry out Sanger sequencing. The conditions followed were the same as explained in the primer design section. The amplicons were purified using a DNA clean & concentrator kit following the manufacturer's instructions. The primer design was carried out as outlined above using Primer Blast and Multiple Primer Analyzer software. The main factor taken into consideration when designing the primers for Sanger sequencing is that the amplicon should start more than 100 bp from the region of interest (SNP) for a correct reading. Primers to produce amplicons ranging from 250 to 600 bp were thus designed. The resulting primers and the expected sequences to be amplified are detailed in Table S3. All the samples were sequenced using the forward primer ( Figure S12). Tables   Table S1. Sequences of the primer sets used in the final assay and the sequences containing the SNP site. (There are two SNP-related specific primers and two other primers (with non-SNP specific terminal bases) used as negative controls.)     * The primer region is underlined and the SNP region is in italics. Figure S1. Schematic representation of the SNP expected in a diploid organism.      (Table S1). β-globin was used as positive control of the reaction.         Table 3. Cases 9, 10 represent cases with a high risk of developing osteoporosis.