Molecular biology & genomics applications are just the beginning


Oligos produced by Enzymatic DNA Synthesis (EDS) have been used in a wide range of molecular biology and genomics applications with comparable functional performance to oligos sourced from commercial suppliers.
SYBR Green®-based qPCR
EDS produces primers that meet the strict quality requirements for SYBR Green-based qPCR

Amplification of DNA or cDNA in the presence of an intercalating dye enables absolute or relative quantification of accumulating reaction products, as well as the amplification template. SYBR Green-based qPCR is a cost-effective and high-throughput method for gene expression studies, genotyping, food safety or pathogen testing, among many other applications. Specificity is a key performance parameter in qPCR, as intercalating dyes impact the efficiency of DNA polymerases, and qPCR readouts (amplification and melt curves) only provide indirect evidence that the reaction product contains the target of interest. Careful primer design, optimized qPCR chemistry and appropriate experimental design (e.g., the inclusion of positive and no-template controls) are critical to ensure low false-positive rates. Primer quality is important to minimize non-specific priming and primer-dimer formation, which decrease amplification efficiency and may skew results.

Amplification of a 200-bp fragment (40% GC) of the P. melaninogenica infA gene using EDS (blue) or commercial (CS, red) primers. DNA was amplified in triplicate from a 10-fold dilution series of P. melaninogenica genomic DNA (300,000 – 30 genome copies per reaction), for 40 cycles using iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories) and a CFX384 Touch Real-Time System (Bio-Rad). The manufacturer’s recommended reaction setup and cycling parameters were followed, except that the annealing temperature was reduced from 60°C to 55°C to accommodate multiple, different primer sets in a single run. Data were analyzed with the CFX Maestro software (Bio-Rad). The reaction efficiency of 90% achieved with EDS primers falls within the acceptable range for qPCR, and was slightly better than the 89% efficiency achieved with commercially sourced primers. Non-specific amplification products were observed in only one of six EDS NTCs, and two of six NTCs with commercial primers (not shown).
End-point PCR
EDS primers support high specificity and reaction efficiency

End-point PCR steps are routinely included in laboratory protocols to (i) confirm the presence of one or more specific DNA fragment(s), and/or (ii) generate a sufficient quantity of material for downstream analysis (e.g. sequencing) or manipulation (e.g. mutagenesis, cloning). Irrespective of the application, high primer quality is needed to achieve the sensitivity, specificity and amplification efficiency required for downstream success.

Virtual gel of human amplicons generated with eighteen primer sets, produced by EDS (E) or sourced from a commercial supplier (C). DNA was amplified from 80 ng of template DNA for 25 cycles with Phire II DNA Polymerase (Thermo Fisher Scientific), using the manufacturer’s recommended reaction setup and cycling parameters. Reaction products were analyzed using a Fragment Analyzer FA 5300 (Agilent Technologies). The dominant band in each reaction product corresponds to the expected amplicon size for each primer set. Slightly faster migration of EDS reaction products results from the presence of 5’-phosphate group in primers produced by default with the SYNTAX System (5’-phosphorylation incurs an additional charge from commercial suppliers). Amplicon purity (% of the total reaction product that corresponds to the DNA fragment of the expected size; reflects specificity) and amplicon concentration (of the desired amplicon only; a measure of reaction efficiency) were subsequently calculated with the ProSize Data Analysis software (Agilent Technologies). In this experiment, EDS primers performed marginally better than commercial primers with respect to both metrics (not shown).
Guide RNA construction for CRISPR/Cas9 gene editing
The SYNTAX System offers benefits for high-throughput sgRNA production by IVT. Guide RNAs can be produced, evaluated, and optimized in rapid cycles of iteration, each only requiring a few days.

The past decade has seen rapid and widespread adoption of CRISPR/Cas9-based methods for genome editing in biological systems ranging from cultured cells to entire animals. The CRISPR/Cas9 system requires two components: a Cas9 nuclease that cleaves the target sequence, and a single guide RNA (sgRNA) that directs the nuclease to the targeted cleavage site. The sgRNA is composed of two parts: a target-specific CRISPR RNA (crRNA) sequence, and a trans-activating crRNA (tracrRNA) scaffold sequence that is common to all sgRNAs. Successful in vivo gene editing depends on delivery of the Cas9/sgRNA complex to the target genome. In vitro assembly of the Cas9-ribonucleoprotein (RNP) complex is gaining popularity as i) it does not require cloning, ii) allows for high-throughput production of fully functional Cas9-RNP complexes that are active upon delivery, and iii) are quickly degraded, thereby reducing the potential for off-target effects. In preliminary proof-of-concept experiments, we have observed no difference in the efficiency of sgRNA IVT production nor the nuclease activity of crRNA oligos produced by EDS vs. those sourced from a commercial supplier.

Electrophoretic assessment of select sgRNAs produced by IVT using EDS or commercial (CS) crRNA oligos. Guide RNAs 1 and 2 target the 3’- and 5’-ends of the KanR cassette of the pET-28(+) plasmid (Novagen), respectively; whereas sgRNA 3 and sgRNA4 target the 3’- and 5’-ends of the lacI cassette. Guide RNAs were designed using the Geneious Prime software. The EnGen® sgRNA Synthesis Kit, S. pyogenes (New England Biolabs) was used for IVT of sgRNAs, which were purified using the Monarch® RNA Cleanup Kit (New England Biolabs). All crRNA oligos were used in combination with the tracrRNA oligo supplied in the EnGen kit, and yielded single-stranded sgRNAs with the expected length of 100 nt. The Control lane contains an sgRNA produced with the control crRNA oligo provided in the EnGen kit. Electrophoresis was performed using denaturing polyacrylamide gels (10% TBE-Urea; Novex) and RNA was visualized with GelRed® stain (Biotium). In subsequent nuclease assays (performed according to manufacturer’s instructions, using the EnGen® sgRNA Synthesis Kit, S. pyogenes; New England Biolabs) sgRNAs produced from crRNAs synthesized by EDS and conventional phosphoramidite chemistry yielded comparable results (not shown).
Variant confirmation
(SARS-CoV-2 spike protein mutagenesis)
EDS primers enable high success rates in multi-site mutagenesis, and produce high-quality Sanger sequencing data

In collaboration with Felix Rey, Structural Virology Lab at the Institut Pasteur, we have evaluated the performance of enzymatically synthesized oligos in site-directed mutagenesis, followed by variant confirmation using Sanger sequencing. The Rey lab studies viruses of global public health and/or veterinary concern. During the COVID-19 pandemic they have focused on SARS-CoV-2 variants of concern—particularly those with diverse mutations in the spike (S) protein, which may be associated with increased transmissibility or immune escape. The SYNTAX System was used to produce (i) primers for use with the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies), (ii) sequencing primers for the confirmation of individual mutations in picked clones, and (iii) primers for tiled sequencing of the entire S gene.

Representative Sanger sequencing results, confirming successful site-directed mutagenesis of ten sites targeted to generate a B.1.1.7 lineage (WHO VoC “alpha”) SARS-CoV-2 spike protein gene starting from the Wuhan Hu-1 S gene sequence. Only three variant sequencing profiles are shown. Mutation N501Y required the use of mutagenesis primers that are challenging to synthesize, even with conventional phosphoramidite chemistry. Results confirm that EDS primers support complex, multi-site mutagenesis as well as high-quality Sanger sequencing, thus allowing a fast and cost-effective workflow for variant gene production.
16S metagenomic sequencing
Primers produced by EDS are suitable for a wide range of amplicon sequencing applications. In metagenomic studies, the SYNTAX System offers unique support for deeper interrogation at a rapid pace.

16S amplicon sequencing offers a high-throughput and cost-effective method for the identification and relative quantification of the archaea and bacteria present in complex microbial populations. The method is based on amplification and sequencing of ≥1 of the 9 variable regions of the ~1.6-kb 16S rRNA gene. To evaluate the performance of EDS primers, the well-characterized, degenerate 515F/806R primer pair was synthesized using the SYNTAX System, or ordered from a commercial supplier. Primers were used to generate V4-V5 amplicons from total DNA extracted from a soil sample collected near the Eiffel Tower in Paris, France. Amplification products were converted to libraries for Illumina® sequencing. Bioinformatic analysis confirmed no significant differences in results obtained with EDS vs. commercial primers.

Genus abundance for libraries produced with EDS and commercial (CS) primers. The twenty most abundant genera identified in library CS1 was used as the basis for calculating the abundance of each genus within the five other libraries. The abundances of all genera outside of the top 20 genera listed on the right were combined and classified as “Other genera” (light green).
Targeted cancer panels
Primers produced with the SYNTAX System are highly suitable for targeted amplicon sequencing. Small panels (up to 48 primer sets per run) can be produced in a standard workday.

Targeted sequencing enables the interrogation of specific genes of interest by NGS. Amplicon-based approaches—in which the regions of interest are amplified by multiplex PCR (in one or multiple pools)—offer streamlined and scalable protocols for library preparation. Despite challenges related to coverage uniformity and duplication rates, deep amplicon sequencing is widely used in cancer research, as input DNA requirements for PCR-based protocols are typically lower than for methods that rely on hybridization capture. In an experiment with a hot spot panel for colorectal cancer genotyping, EDS primers performed comparably or better than primers from two commercial suppliers in terms of overall sequencing data quality, amplicon uniformity and variant calling results. In addition, the system enables on-demand synthesis of primers that may be needed to optimize panel performance (amplicon coverage) or add additional content.

Identification of genetic variants in amplicon libraries prepared with EDS and commercial (CS1 and CS2) primers. SNP analysis was performed using the Geneious software. The relative abundance of specific SNPs associated with two tumor suppressor genes (APC and TP53) are shown. The HT-29 cell line is homozygous for the SNPs in amplicons 5 (G > A) and 37 (C > T), and heterozygous for the amplicon 7 SNP (A insertion).
SARS-CoV-2 sequencing (ARTIC protocol)
ARTIC V3 primers synthesized by EDS perform comparably to commercial primers with respect to coverage uniformity and variant calling, and support phylogenetic analysis of clinical isolates

In collaboration with The Jackson Laboratory for Genomic Medicine, the University of Zacatecas Molecular Medicine Laboratory and the Public Health Laboratory in Zacatecas, Mexico, we compared the performance of the ARTIC V3 panel (218 primers) produced either by EDS or ordered from a commercial supplier. Libraries were generated from two synthetic SARS-CoV-2 RNA controls (10,000 genome copy equivalents per reaction), as well as five clinical samples (with RT-qPCR Cq values ranging between 18.5 and 30.9). Sequencing data for libraries generated with EDS and commercial primers showed no significant difference respect to coverage uniformity and variant calling (not shown).

Phylogenetic analysis of four Mexican SARS-CoV-2 isolates with RT-qPCR Cq values <30, in the context of 192 SARS-CoV-2 sequences collected from different geographical locations between December 2019 and June 2020. Of these, 146 sequences were retrieved from the ncov Nextstrain database (https://github.com/nextstrain/ncov), whereas sequences for other Mexican isolates (n = 46) were obtained from the NBCI database (https://www.ncbi.nlm.nih.gov/genome). The four sequences from this study clustered together in clade 20A, which is the predominant clade based on the genomes from the Nextstrain database. Mx 18.5 and Mx 28.6 clustered more closely together (clade 20B), while Mx 25.6 was the most divergent (clade 20C). Analysis was performed using the Nextstrain command-line interface (https://github.com/nextstrain), and results were visualized on the Auspice platform (https://auspice.us).