What’s that Organism? Using DNA Barcoding for Species Identification

In both the lab and field, it is important to know what species we are working with. While morphological data has always been a tried and true method of identifying species, DNA barcoding allows us to identify species when we don’t have that option (e.g. if we don’t have enough of a specimen to identify by morphology, or when the morphological differences are so minute that the specimen is nearly impossible to identify visually).

Even in cases where visible morphological differences are present, DNA barcoding can still serve as a powerful tool. When you work with fungi in the lab like I do, two specimens of the same species can sometimes look completely different from each other— this is sometimes an effect of age, growth conditions, or a variety of other factors. Now, I’m not a professional fungi taxonomist, so DNA barcoding is an alternative yet effective method of confirming species identification.

DNA barcoding is the practice of obtaining and sequencing a particular section of DNA that, once compared to the millions of sequences in online DNA sequence databases, is able to help us determine what species the DNA belongs to.

So, you have a biological sample from an unknown organism— how do you go from this sample to an identified species? With this quick-start guide, you’ll be ID-ing species in no time.

Step 1: DNA Extraction

Whether you are working with plants, fungi, animals, or something else entirely, each type of organism comes with its own DNA extraction challenges. For example, many plants have secondary metabolites that can interfere with the extraction, while fungi have tough chitinous walls that are difficult to break down. Some kits that I use to overcome these challenges include the Yeastar™ Genomic DNA Kit (from Zymo Research) for fungal matter, or the DNeasy® Plant Mini Kit (from QIAGEN®) for plant matter.

Step 2: Choosing Your Region of Interest and Amplifying DNA

Depending on the type of organism you are working with, many scientists have come to the consensus of using particular regions that are effective at discriminating samples to the species level. These regions are known as universal barcoding regions.

For animals, the universal barcoding region is the cytochrome c oxidase subunit 1 (cox1 or CO1).¹ For plants, we often target the internal transcribed spacer 2 (ITS2) region and the large subunit of the Ribulose bisphosphate carboxylase (RbcL) gene.² For fungi, we target the internal transcribed spacer (ITS) region.³

That being said, the DNA sequences of certain taxa are very highly conserved, meaning that sequencing the universal barcoding regions will not give enough clarity to differentiate certain species. When this happens, you may need to head to current literature to search for additional, less highly conserved regions to sequence to obtain better species clarity.

Now that you have identified the regions of DNA that you want to amplify, you are ready to design primers that will target your desired region. From there, you can run a PCR to amplify your DNA.

Step 3: Sequencing and Identification

Now you have some freshly amplified DNA, you can perform a sequencing reaction on those strands of DNA. Once you have your DNA sequences, you are ready to analyze your sequences, then head over to NCBI’s BLAST (Basic Local Alignment Search Tool). BLAST is a program that will compare the sequence that you feed it against a database of DNA sequences, aligning your sequence to its closest matches and ranking them by similarity. This allows you to determine what your unidentified organism is!

Whether you decide to use barcoding to identify a new species of insect in the Amazon rainforest or to identify the bacteria contaminating your experiments, barcoding serves as a useful tool to expand our knowledge of the diversity around us. Happy identifying!

References:

1. Hebert PDN et al. (2003) Biological identifications through DNA barcodes. Proceedings: Biological Sciences 270 (1512):313-321.
2. Yao H et al. (2010) Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals. Plos One https://doi.org/10.1371/journal.pone.0013102
3. Schoch C et al. (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. PNAS 109 (16):6241-6246.