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Why DNA sequences rather than qPCR?

Our blue print (DNA) is a remarkably stable molecule. So much so, that traces of it can persist upwards of a million years in the right circumstances. This is something we are involved at the forefront of studying producing world-class research (Kistler et al 2017). For this reason eDNA has become increasingly used in the realm of conservation. Indeed, in our research we have found eDNA and been able to reconstruct environments from Mesolithic Britain, again at the forefront of the field (Smith et al. 2015), and our research continues to reconstruct the palaeolandscapes in the sea surrounding the UK, from DNA sequences (Lost Frontiers). We are also at the forefront of reconstructing entire genomes from thousands of years ago (Kistler et al. 2018). We track the minutest traces of DNA through time that can be found.

So why use DNA sequences? DNA is a code, it is also a unique identifier. You cannot be sure of the origin of the DNA you have, until you read the DNA sequence, only at that point do you have a proof. There is no more accurate way of knowing what organism you are dealing with than reading its DNA sequence.

There are, however, numerous ways in which DNA which has been specifically amplified from an environment might not be the organism you expected it to be. Proxies of DNA sequence, such as used with qPCR, can and in our experience do show spurious signal, especially from uncontrolled natural environments and also where the DNA is degraded. There is also some misinformation out there regarding the sensitivity of qPCR over normal PCR - the former cannot detect a signal from fewer template molecules than the latter, if anything the reverse is true. This is because the former involves chemically modifying primers, the little anchors that attach to DNA templates, which in turn affects their affinity (for the worse). If this notion were true, then you might observe the method being used in the field of ancient DNA. For this reason we refuse to use qPCR approaches in analyses, and indeed have advised NGOs to do likewise in the past.

In short, obtaining DNA sequences is the most sensitive and accurate way of measuring the presence of organisms in the environment.