Genaphora’s platform is based on two proprietary principal developments:
1. Computational-biology platform
Genaphora developed its own computational biology platform. Using thermodynamics calculation and modifications of linguistic complexity algorithms enables the searching for the best fit primers and probes in whole genome sequence of any given organism. Accurate design of the primers and probes as well as choosing the best target sequence are crucial and critical steps for higher standard real-time PCR assays.
2. Chimeric DNA/RNA primers and probes Chimeric
DNA/RNA sequences1 – (patent registered in the USA2 and China3 and pending worldwide) enables significant increase in sensitivity and specificity of the singleplex and multiplex real-time qPCR assays. During DNA amplification, target sequences are specifically amplified, while primer dimers and other nonspecific sequences are eliminated. This results in a highly sensitive assay down to single cell. Genaphora proved the advantage of chimeric primers technology in non-specific (SyBR green) and specific probes qPCR assays. Harnessing the above two elements together have enabled the company to produce its high quality primers and probes addressing a fundamental problem in real-time qPCR assays: the almost inevitable formation of primer dimers. Primer dimers are non-specific by-products of the qPCR reaction. The formation of nonspecific products elevates the assay detection threshold (thereby reducing the assay sensitivity), resulting in some cases, false negative answers. The most common solution to primer dimers is sequence specific probes, designed to detect only the desired PCR product. However, although most of the probes are sequence-specific, none of them prevents the formation of primer dimmers. Hence these sequence specific probes do not avert their negative effect on the assay’s sensitivity, accuracy and efficiency, but rather cover up and hide them. Genaphora’s technology postpones and practically eliminates the formation of nonspecific byproducts, thus improving assay sensitivity. Combined with a high resolution melt analysis (HRM) using non-specific dyes, Genaphora’s technology could even de-necessitate the use of expensive sequence specific probes.
The advantage of DNA/RNA chimeric primers is demonstrated in the following figure.
Data graph, Tm plot and standard plot of qPCR reaction using DNA primers and chimeric primers. The linearity range of the chimeric primers data graph is wider than that of the DNA primers, resulting in higher sensitivity. Tm analysis of the DNA primers demonstrated peaks of nonspecific products at high dilutions, while in the case of the chimeric primers, the only significant peak was that for the target gene amplicon. The use of chimeric primers practically eliminated the nonspecific side products (“noise”) and thereby, increases the assay’s sensitivity.
Linearity and maximum sensitivity down to single copy
The molecular weight of the whole genome of Cryptosporidium parvum was calculated. A known concentration of Cryptosporidium Genomic DNA was received from ATCC. The exact copy number of genomic DNA copies was calculated and sample duplicates were diluted, in serial decimal dilutions, down to single copy per sample. Each sample was tested in a real time qPCR assay.
This bar chart of the delta Cq values demonstrates a linearity of at least 7-orders-of-magnitude and limit of detection of only a few DNA copies. The standard plot shoes an efficiency of 100%.