In this article, Jim Campbell, founder of SureScreen Scientifics, introduces the science behind eDNA analysis and its application in ecology…
Britain has played a major role in the science behind the so-called ‘blueprint of life’, DNA, or to give its full chemical name, deoxy-ribonucleic acid. In 1859 Darwin’s ‘Origin of the Species’ gave an insight into the consequences of subtle variations that produce offspring with slightly different characteristics to that of the parents, bringing with it superior or inferior chances of adaptation and survival. Darwin’s own life may have been influenced by his own genetics and upbringing; he was the grandson of the eminent and outspoken physician Erasmus Darwin and the entrepreneur, philanthropist and pottery king Josiah Wedgewood, both of whom encouraged him in his studies at Edinburgh and Cambridge.
In 1902, Sir Archibald Edward Garratt, practicing in Oxford, was the first doctor to connect genetic traits to particular diseases, then in 1953, Rosalind Franklin’s x-ray imaging at Cambridge resulted in Crick and Watson’s famous DNA model. Modern applications of DNA analysis began in the 1970’s when Frederick Sanger, also at Cambridge, developed methods of genetic sequencing using principles we still use today. The modern process of DNA profiling was developed in 1984 by Sir Alec Jeffreys at the University of Leicester. But it wasn’t till 1995 that the Forensic Science Laboratories used mass DNA profiling in a murder case.
Since those early days, computerised analysis and better equipment has allowed DNA analysis to become cost-effective in areas outside criminal work, allowing ecologists to use DNA to identify the presence or absence of species, adopt early detection and monitoring of invasive species, gather data on genetic biodiversity within a species or family group, and even uniquely identify individuals of a species.
DNA – The ultimate chemical photocopier
So, what is DNA? Put very simply, it is a giant molecule consisting of a spiral staircase with sides made of sugars and phosphates, on which the rungs are assembled from four simple purine and pyrimidine nucleotides Adenine (A) and Thymine (T) or Cytosine (C) and Guanine (G). Since A only binds with T, and C only with G, the DNA in the cell replicates by splitting into two along its length with the help of enzymes, thus exposing the unique array of chemical ‘rungs’ onto which the corresponding nucleotides can assemble to produce an exact duplicate of the original. It happens about 50 billion times a day in your own body as cells wear out and get replaced.
It’s hard to think of anything more complicated than DNA. Consisting typically of three billion pieces of genetic code, if you could stretch it into one strand, the DNA in every one of your cells would stretch about 2 metres, but nature packs it into tight coils so it occupies only a corner of the nucleus in each cell. And because your body contains around 6 billion cells, if it was possible to straighten DNA out and join all your DNA into a single strand it would stretch across the width of the solar system twice. It’s a bizarre fact, but theoretically possible because that strand is only two nanometres thick. None of this, or indeed life itself would be possible if DNA wasn’t helical, because both the sugars and the phosphates that form the spiral staircase are water soluble. That spiral curve binds the sugar and phosphate molecules together at just the right angle to prevent water molecules getting in and dissolving them and the bases are hydrophobic and repel water.
But just like an ageing photocopier with a scratched screen, as your cells replicate and get replaced, errors can creep in over time, with misplaced pieces of code resulting in polymorphisms and variations. Polymorphisms (from the Greek poly (meaning multiple) and morph (meaning form), describe multiple forms of a single gene that exists in an individual or among a group of individuals, and are common in every species. Examples include whether you have attached or detached earlobes, or what blood type you have. On the other hand, variations or ‘mutations’ (from the Latin ‘mutare’ to change) are small defects that develop, often due to virus attacks, u/v light damage, radiation, chemicals and mistakes during replication, which go on to produce subtle changes in future generations. This effect can result in genetic drift, in which allele frequency changes with time, usually with inferior traits disappearing due to competition and environmental pressures. This effect is precisely what Darwin documented so meticulously in his famous studies.
As a consequence, each species has its own genetic code, each family member has characteristic genetic similarities, and each individual has its own DNA signature which it leaves behind wherever it has been. These genetic variations are what allow us to analyse a piece of animal scat and identify the species, or even test pond water for each and every individual species of newt that lives there. Environmental DNA analysis (eDNA) is now very popular for Great Crested Newt (GCN) monitoring as it is easier, more accurate, less invasive and is more cost effective.
Much of the DNA in an organism is common across the whole species range. Humans share 98.4% of their DNA with Chimpanzees, and anecdotally around 60% with the banana. Here, we need to make a key distinction. In eDNA we are looking for species-specific bits of DNA that are common to all members of that species, and no other. This is very different to crime scene DNA work where we are looking for DNA common only to that one individual. Criminal DNA work looks for unique bits of DNA that has become corrupted in the copying process, to make that individual unique; single-nucleotide polymorphisms (called SNP’s and often referred to as ’snips’). These are the bits of DNA that identify an individual in the crowd.
So, looking for criminals, we’re searching for those unique snips whereas when looking for GCN we look for species DNA that is common to all GCN’s. We could even look for DNA specific to the banana within human DNA, but we see that as a fruitless exercise.
The process of analysing DNA
Designing an assay for a new target species starts by reviewing genetic sequences to find a portion that is unique to that species. Luckily, most common species have already been sequenced and the data is available to molecular biologists in bioinformatics databases. Using that unique sequence, we can manufacture primers, small sections of DNA that bind to specific spots at the beginning and end of a short segment of DNA. This segment is conserved across all individuals of that specific species, but is different from equivalent segments in other species. We can either choose specific sequences to test for, say, all newts, or be more specific and test for only great crested newts.
In order to analyse DNA, we use quantitative polymerase chain reaction (qPCR) or conventional PCR. This process involves mixing extracted DNA with primers and other chemicals which then undergo a three-step thermal cycling process. The first step, denaturation, occurs when the reaction mixture is heated to 95 degrees Celsius. The hydrogen bonds between bases are broken and the DNA helix separates into two single-stranded molecular chains. Next, the temperature is lowered to around 55 degrees Celsius and the primers anneal, or form hydrogen bonds, to their complimentary bases as designed. Elongation of the DNA molecules occurs when the temperature is heated back to 72 degrees Celsius, and a polymerase enzyme incorporates matching nucleotides from the solution into the newly-forming double-stranded DNA molecule. Each round of thermal cycling results in a doubling of the number of DNA copies.
The qPCR technique uses the same basic thermal cycling steps as conventional PCR, but includes the addition of a probe. Probes are designed to align within the amplified region, and contain fluorophore molecules. The probe will anneal to the single-stranded DNA molecule at the same time as the primers. When the enzyme elongates the molecular string, the probe will fluoresce, or emit small amounts of light at a measurable wavelength. These light emissions are measured by the thermal-cycling machine. As more and more copies of DNA are produced, more and more light will be detected. The rate of amplification depends on the starting concentration of molecules. So, if there is no GCN DNA present to start with, there will be no amplification. We add a synthetic DNA sample to the sample kits when we manufacture them, and we analyse that at the same time as the GCN to show the sample had not deteriorated, and that our extraction process was successful. This synthetic DNA spike also identifies any chemicals in the water that would inhibit DNA or extraction, if they are detected, we will ask for a fresh sample.
The GCN monitoring and analysis that takes place for Natural England’s planning procedures is a good application of eDNA. Sampling and testing must be carried out in strict accordance with the directives laid down in document WC1067. This requires up to 20 sample locations from around the pond, concentrating on areas where GCN are likely to congregate or enter and exit the pond. This is collected into a whirlpack bag, well mixed, and then pipetted into six tubes containing DNA preservative. As we have explained, DNA species analysis is so sensitive that the quality of collection is critical to avoid cross contamination from other ponds. Ecologists should avoid entering the water, risking transfer of DNA, or stirring up mud from the bottom of the pond as this could still harbour ancient GCN DNA from earlier seasons. Ladles and bags from collection kits should not be reused at other sites. Used equipment should be sent back with the samples as the contents are then recycled responsibly.
Opportunities for Ecologists
A recent example of eDNA expansion is SureScreen’s work on the white-clawed crayfish (Austropotamobius pallipes), the only freshwater crayfish species indigenous to the United Kingdom and until recently, common across an extensive range over most of England and Wales. Over the last 40 years its population numbers have seen a dramatic decline, subsequently becoming listed as endangered on the IUCN Red List. One of the main reasons attributed to this decline has been the introduction of non-indigenous crayfish species, for commercial purposes during the 1970’s. These species include the signal crayfish (Pacifastacus leniusculus), a more dominant species and a carrier of the crayfish plague (Aphanomyces astaci), a water mould which has little effect to the invasive species, yet can have a devastating impact on the native A. pallipes, often completely wiping out populations.
Crayfish, particularly at low abundance, can be notably difficult to find using existing survey efforts. This makes current survey techniques expensive and time exhaustive, often resulting in small pockets of isolated data with little large-scale implication. After two years of eDNA study on the River Ecclesbourne as part of the method development, we can now analyse the DNA of the white clawed crayfish, and other crayfish species, as well as crayfish plague from a water sample. One outcome of our work has been to identify a critical role that a particular weir has on segregating signal crayfish and crayfish plague along the river, showing how e-DNA mapping can revolutionise the maintenance of our waterways to optimise diversity.
eDNA analysis offers the ecologist exciting opportunities to provide better analyses that are more comprehensive, more accurate, and with less-invasive techniques. It also provides areas for business expansion. The public response to environmental pressures highlighted by popular TV programmes like Blue Planet 2 has been very encouraging. The UK government has committed to continue spending on the environment and has many initiatives if you know where to look. Currently, restrictions on importing plastic waste into China is also leading to increasing awareness of pollution, as Britain looks to treat much more of its rubbish locally.
From the recent horsemeat scandal to the spread of disease-carrying mosquitos, DNA analysis provides the ecologist with a fantastic tool for species identity and tracking. Wildlife crime alone has become a major area for ecologists armed with the latest eDNA technology in their toolkit, with CITES regulating the trade in more than 35,000 species, and millions more that are endangered but not covered by the convention. The beauty of this technology is that they are all are identifiable and trackable by their DNA.
About the Author: Jim Campbell began his career in 1968 with the Home Office Forensic Science Service where he developed hands-on knowledge of most analytical techniques, mainly dealing with criminalistics and toxicology. He gained an honours degree in materials in 1974. In 1991, Jim started SureScreen to apply forensic techniques to commercial problems, and soon began working on unusual DNA applications connected to fraud, damage and personnel tracking. His career has included the training of most European police forces in drug detection methods, advisor to DRUID (DRivers Under the Influence of Drink), TISPOL, and the development of methods for hair testing for drugs, and latterly for alcohol too. In the early years of SureScreen Jim was responsible for developing accurate, low-cost rapid tests for healthcare applications. SureScreen participated in the Q-Cancer project and was lead organisation in the RESPOC project, both of which provided proof of principle for rapid DNA testing of tumour tissue and tuberculosis/pneumonia respectively. More recently, DNA analysis has featured heavily in his workload, including the development of a test for missing persons in watercourses, canals and lakes. He remains a committed forensic scientist/researcher, pushing technology into fields that benefit from the huge investments already made on the methods in other areas. eDNA benefits hugely from the complex technology and equipment originally designed at great cost to catch criminals.