Through the re-tooling of a best-on-market extraction protocol, our system both maximizes and guarantees yield from bivalve larvae. Other services utilize generic DNA extraction kits, or home brew isolation methods, that cannot mitigate inhibitors of PCR or reliably break down calcium carbonate shells of larvae in late-stage development.
Our services differ in three ways
1) the use of a PCR-inhibitor minimizing chemistry that sequesters and removes inhibitors to maximize yield and quality
2) a modified homogenization protocol that specifically targets and maximizes recovery from bivalve larvae at all stages and
3) Eluate buffering conditions designed for long-term sample storage with high preservation fidelity.
Our unique probe design allows for the most sensitive assay available. Other services utilize either SYBR green or Taqman systems. SYBR green indiscriminately binds to any double-stranded DNA resulting in extremely high background noise and, therefore, a small usable signal range. Taqman systems typically involve the use of two fluorescent molecules: a reporter and a quencher. However, due to overlapping fluorescence spectra of the reporter and quencher, this design leads to both background noise from the quencher and the inability to use multiple probes together. Our probes have been specifically designed to avoid these common issues by using a primer/probe set with dual quenching. Quenchers have no native fluorescent signal, thereby eliminating baseline noise. Our primer/probe dye design allows for not only a superiorly sensitive assay but also the ability to multiplex at least five assays per reaction.
Aside from designing a highly sensitive probe, we have also designed a method for selecting probe binding regions that yield species- and gene-specific results. Other services using SYBR green or traditional Taqman assays struggle with environmental sampling and partially degraded samples. SYBR green requires amplicons that are larger than 600 bp for optimal signal, however, this becomes highly problematic as the longer the strand, the more likely it is to be affected by degradation, and the longer the reaction, the more likely the enzyme will be inhibited and lose efficiency. Traditional Taqman probes use an algorithm in which specificity is to the gene more than to the organism leading to a lot of trial and error to prove species specificity. Our primer/probe sets are designed by using a proprietary species specificity algorithm and a modified version of the leading primer/probe design parameters for gene specificity and robust signal. The resulting sequences for the primer / probe sets are highly sensitive, efficient, species and gene specific, and proprietary.
Instead of using a Taq enzyme master mix formulated for in vitro research, we use a master mix chemistry specially formulated for environmental DNA and our probes are stored in buffers that maintain this proprietary formulation. The polymerase master mix we use contains stabilizers to reduce the effect of any inhibitors in the PCR reaction, including specialized buffering conditions and biologics that protect the enzyme from inhibitors by blocking their binding sites. With this system, along with the inhibitor minimization chemistry used during DNA extraction, we can ensure 100% enzymatic efficiency with any environmental sample allowing for clear, repeatable, and trustworthy results every time.