2. Methods

MSD Technology:
MSD’s electrochemiluminescence detection technology uses SULFO-TAG labels that emit light upon electrochemical stimulation initiated at the electrode surfaces of MULTI-ARRAY and MULTI-SPOT microplates.

Electrochemiluminescence Technology

• Minimal non-specific background and strong responses to analyte yield high signal-to-background ratios.
• The stimulation mechanism (electricity) is decoupled from the response (light signal), minimizing matrix interference.
• Only labels bound near the electrode surface
• are excited, enabling non-washed assays. Labels are stable, non-radioactive, and directly conjugated to biological molecules. Emission at ~620 nm eliminates problems with color quenching.
• Multiple rounds of label excitation and emission enhance light levels and improve sensitivity.
• Carbon electrode surface has 10X greater binding capacity than polystyrene wells.
• Surface coatings can be customized.

SNP Detection with OLA/Single Ligation and N-PLEX:
The oligonucleotide ligation assay (OLA) and single ligation used probes that are specific for DNA sequences upstream and downstream of the SNP of interest, with only exact matches at the SNP site allowing for the ligation of the two probes. The ligated probes were hybridized to spot-specific capture oligos on the N-PLEX plates to allow for detection.

SNP Detection

• Each well in an N-PLEX 96-well plate has 10 unique capture oligos attached to the surface of the plate, allowing for the detection of up to 5 SNPs per well.
• Three probes were needed per target: a biotinylated
• (downstream) probe and two (upstream) probes that recognized either polymorphic base and contain a sequence complementary to a specific capture oligo.
• Probe characteristics: 13-18 nucleotides in length; probe ligation temperature between 64-67°C for a given pair.
• DNA ligase was used to join upstream and downstream probes that aligned correctly on a given DNA sample. Fragments of unmodified template complements were added to prevent bridging of nonligated probes.
• OLA/single-ligation products (from synthetic oligos or PCR products from DNA samples) were hybridized to the appropriate capture oligo on the N-PLEX plate, bound by SULFO-TAG labeled streptavidin, and analyzed using an MSD® instrument.
• Synthetic oligos were obtained from IDT.
• DNA was extracted from HL-60 cells using the PureGene Cell and Tissue Kit.
• Blood samples were obtained from BioIVT, and DNA was extracted using the MagMAX DNA Multi-Sample Ultra 2.0 Kit.
• PCR was conducted with site-specific primers using the MyTaq HS Mix or Platinum II Taq Hot Start DNA Polymerase (fast PCR mix), according to manufacturer recommendations.

3. Selected SNPs

Two SNPs, rs7412 and rs429358, in the APOE gene shown to be genetic risk factors of Alzheimer’s disease (AD) were chosen for the study.

4. Results – Assay Design and Optimization

Optimized Protocol

1. Perform fast PCR on extracted samples with site-specific primers and DNA polymerase (2 min at 95°C and 35 cycles of 15 sec at 95°C, 15 sec at 60°C, and 15 sec at 72°C).
2. Perform single ligation on PCR product with multiplexed probe sets and DNA ligase (2 min at 95°C and 15 min at 65°C). Block N-PLEX plate for 15 min at 37°C.
3. Wash plate and add ligation product in hybridization buffer (50 µL per well) to plate. Incubate for 30 min at 37°C.
4. Wash plate and add detection solution (50 µL per well). Incubate 15 min at room temperature.
5. Wash plate and add read buffer (150 µL per well). Analyze with MSD instrument.

Synthetic oligo templates show specificity for the appropriate allele using the optimized conditions of no ligation cycling and a ligation temperature of 65°C. Both SNP assays fit within our allele frequency guidelines for calling SNPs: Homozygous ≥80%, Heterozygous =30-70%, Not Present ≤20%.

5. Results – Sample Testing

Genomic DNA was extracted from HL-60 cells and 79 human whole blood samples and amplified with site-specific primers via PCR. The optimized protocol was used to run a single ligation and fast N-PLEX on the two target SNPs in multiplex.
The APOE allele and genotype frequencies in the sample set are similar to those reported from population studies in 11 countries. The majority of the samples (45/79) have the ε3/ε3 genotype. There is one sample with ε4/ε4 (major alleles for rs7412 and minor alleles for rs429358). None of the 79 samples has the rarest (ε2/ε2) genotype. All samples fit in our allele frequency guidelines for SNP calling.

6. Results – Genotype Confirmation via Sanger Sequencing

POE PCR reactions for all 79 samples were loaded and run on 2% agarose gels. The desired fragment was excised and purified using the QIAquick Gel Extraction Kit. The purified PCR products, along with the forward primer, were used for Sanger sequencing.
There was 100% concordance in genotyping results between N-PLEX and Sanger sequencing for APOE rs7412 and rs429358.

7. Conclusions

This report highlights a novel method to quickly identify APOE genotypes. The assay improves sample throughput, making DOWNLOAD POSTER this a viable alternative to sequencing and other methods. Lastly, this method can be easily adapted to target SNPs or
insertions/deletions that are identified as risk factors for other diseases.