“A DNA collected from varieties of environmental samples such as water, soil, animal waste or even from the air is called environmental DNA or eDNA.”
DNA is a building block present in all living forms on earth with a similar chemical structure having deoxy sugar, phosphate and nitrogenous bases.
However, the order of nucleotides is different in a different organism which makes them different.
DNA can remain stable for thousands of years. For example, some of the DNA samples collected from the permafrost are of thousand years old.
However, some biotic or abiotic stress can degrade environmental DNA samples.
All living organisms of earth shade off some amount of DNA or genetic material in their surrounding environment in the form of urine, blood, faeces, gametes, mucus or any biological fluid bodily. Even the decomposed body is one of the rich sources of eDNA.
Although the source of the organism is unknown to scientists, they use these samples for analysing its present and past situations.
The sample might be heterogenous contains nuclear DNA, mitochondrial DNA or even chloroplast DNA in case of plants.
However, in eDNA analysis, the mitochondrial DNA is used for the identification of the species.
There are several benefits of using it.
Only a single copy of nuclear DNA is present in a cell while thousands of copies of mtDNA are are present in the cytoplasmic organelle bounded mitochondria.
In addition to this, the mtDNA is diverse enough to identify every organism. Read more on organelle DNA: Extrachromosomal inheritance class 2: organelle DNA.
Process of environmental DNA analysis:
The environmental sample proceeds in the following steps
- Sample collection
- DNA extraction
- DNA purification
- DNA amplification and quantification
- DNA sequencing
Sample collections:
Seawater, pond water, soil, sediments or surface swab are taken as a source of sample for DNA analysis.
Since the DNA remains stable for thousands of year, no need to store sample in a strict sample collection conditions. The sample is then sent to the laboratory for DNA extraction.
DNA extraction:
The sample contains the DNA of many organisms, we need to first extract DNA from the sample.
Depending upon the type of sample and the aim of the researcher, the DNA extraction method varies.
For example, if someone wanted to study the trace of plant DNA in any biological sample then he must have to use the CTAB plant DNA extraction method.
On the other side, if someone wishes to do metagenomic analysis for some pathogen, he needs a kit for pathogen or bacterial DNA extraction.
Several common DNA extraction methods used for environmental DNA analysis are:
- Phenol-chloroform DNA extraction method
- Proteinase K DNA extraction method
- CTAB DNA extraction method
However, I strongly recommended ready to use environmental DNA extraction kits.
DNA purification:
Contaminants may inhibit PCR amplification, thus it is very important to remove contaminant before doing PCR.
For example, polyphenolic compounds frequently found in the plant samples, the humic acid is present in almost all types of soil sample.
In addition to this, chemicals used during DNA extraction, all these factors collectively inhibit PCR amplification. We need to remove it.
The alcohol purification method is widely used traditional DNA purification method used during DNA extraction.
However, our sample is precious, one must use the ready to use DNA purification kit.
After DNA purification, check DNA purity and quantity before proceeding further.
The pure DNA must have purity nearly by ~1.80 and quantity more than 50ng.
After DNA purification, the sample is processed for DNA amplification and quantification.
Related article:
PCR:
Using the sequence-specific primers, one can amplify as well as quantify the DNA present in the sample.
The Taq DNA polymerase amplifies the DNA and generates millions of copies of DNA while the fluorescent dye or hybridization probe present in the sample, quantify the DNA by binding with it.
A significant amount of amplification using the specific set of primer consider as positive for that particular species. Further to this, random primers can also be used for whole taxa.
Related article:
DNA sequencing:
If the DNA sample is unknown or we want to find out some unknown DNA present in the sample, the DNA sequencing is the best method.
In the DNA sequencing, the sequence of the nucleotides present in a DNA sample is determined using the fluorescent chemistry.
By using the computational and bioinformatics tools the determined sequence can be identified by comparing it with the reference sequences.
Related article: DNA sequencing methods.
This process is called as metabarcoding which is used in the biodiversity studies. The entire process of the analysing the environmental sample is called s metagenomic analysis.
In the metabarcoding method the repetitive DNA regions from the mtDNA, cpDNA or ribosomal DNA genes are amplified, quantified and sequenced for creating a barcode for particular species of taxons.
Sedimentary ancient DNA:
The sediments of DNA of different mammals, insects, prokaryotes any other organism present in ancient sediments are called sedimentary ancient DNA.
The presence of absence of one particular organism at a particular place in ancient times can be identified using the sedimentary ancient DNA.
The PCR can be done either for single species what we want to study using the species-specific primers or by multiple sets of primers for studying many organisms at once.
Related article: Multiplex PCR.
Using the state of the art next-generation DNA sequencing methods many environmental DNA samples sequenced in one reaction by limiting efforts and cost.
Advantages of environmental DNA:
Cost-effective: the eDNA analysis method is very cheap as compared with the traditional survey-based methods. Only one person can collect a sample in a tube or collection bottle and send it to the laboratory.
Stressless: the sample can be collected without giving stress or disturbing the habitat or the lifestyle of organisms. The sample collection method for eDNA analysis is non-invasive.
Highly accurate: the method is entirely based on the DNA analysis thus it is more accurate and precise.
Applications of environmental DNA:
The eDNA is used in the conservation monitoring of different species, subsequently, DNA barcoding can be done.
eDNA based metagenomic analyses help in identification of many fungi, mussels, fishes, plants, animals and pathogens from the unknown sample.
Using eDNA method, one can identify species which are at the risk of extinction, also, the location of invasive and keystone species from the population can be identified.
We had covered an amazing article on how to identify the location of an animal and their migration route using the eDNA method, read it here: Can we track animal migration by eDNA?
For the lesser-known, rare aquatic or other species, biodiversity can be measured accurately and precise information about their habitat can be estimated.
Species distribution and relative abundance can be studied using the eDNA method.
Prevalence of specific species in the environment can also be determined.
Besides, the growth of the endangered species can be determined, whether it is increases or decreases.
Detection of pathogens can also be done using the quantitative PCR based eDNA analysis.
The parasites cause swimmers itching are now detected using this method from the lakes, ponds or swimming pools.
Information: as per the recent environmental DNA study, Greenland was a forest before 2 million years ago.
Challenges in environmental DNA analysis:
Contamination:
One of the greatest limitations of any PCR assay is either contamination or cross-contamination of a sample with other DNA.
Once the sample contaminated the chance of false-positive results increases which leads to misinterpretation of results and wrong identification of species.
The contaminant can be other samples or other DNA or pathogens.
Inhibitors:
Some chemicals are strong PCR inhibitors. The humic substance present in the eDNA sample is one of them which inhibits the activity of Taq DNA polymerase and results in reaction failure.
(Note: our Taq DNA polymerase is an enzyme which synthesises the growing DNA strand by incorporating dNTPs on it).
Sediment particle present in water or other chemicals used during DNA extraction are also inhibits PCR amplification.
Conclusion:
Estimating biodiversity is a time-consuming process, using the molecular genetic technique alike PCR or DNA sequencing it can be done within an hour. Also, the accuracy of the results is higher as compared with the manual methods. Analysis of environmental DNA is one of the most innovative applications of PCR.
Sources:
- Thomsen PF & Willerslev E. “Environmental DNA- An emerging tool in conservation for monitoring past and present biodiversity.” Biological Conservation. 2015 (183); 4-18.
- Peters L, Spatharis S, Dario MA, et al. Environmental DNA: A New Low-Cost Monitoring Tool for Pathogens in Salmonid Aquaculture. Front Microbiol. 2018;9:3009. Published 2018 Dec 7.
