01/03/2017 DNA Learning Center Barcoding 101

Laboratory (/lab/)

Using DNA Barcodes to Identify and Classify Living Things:

INTRODUCTION

OBJECTIVES

The Using DNA Barcodes to Identify and Classify Living Things laboratory demonstrates several important concepts of modern biology. During the course of this
laboratory, you will:

Collect and analyze sequence data from plants, fungi, or animals – or products made from them.
Use DNA sequence to identify species.
Explore relationships between species.

In addition, the laboratory experiment utilizes several experimental and bioinformatics methods in modern biological research. You will:

Collect plants, animals, or products in your local environment or neighborhood.

INTRODUCTION

Taxonomy, the science of classifying living things according to shared features, has always been a part of human society. Carl Linneas formalized biological
classi cation with his system of binomial nomenclature that assigns each organism a genus and species name.

Identifying organisms has grown in importance as we monitor the biological e ects of global climate change and attempt to preserve species diversity in the face of
accelerating habitat destruction. We know very little about the diversity of plants and animals – let alone microbes – living in many unique ecosystems on earth. Less
than two million of the estimated 5-50 million plant and animal species have been identi ed. Scientists agree that the yearly rate of extinction has increased from about
one species per million to 100-1,000 per million. This means that thousands of plants and animals are lost each year. Most of these have not yet been identi ed.

Classical taxonomy falls short in this race to catalog biological diversity before it disappears. Specimens must be carefully collected and handled to preserve their
distinguishing features. Di erentiating subtle anatomical di erences between closely related species requires the subjective judgment of a highly trained specialist –
and few are being produced in colleges today.

Now, DNA barcodes allow non-experts to objectively identify species – even from small, damaged, or industrially processed material. Just as the unique pattern of
bars in a universal product code (UPC) identi es each consumer product, a “DNA barcode” is a unique pattern of DNA sequence that identi es each living thing. Short
DNA barcodes, about 700 nucleotides in length, can be quickly processed from thousands of specimens and unambiguously analyzed by computer programs.

The International Barcode of Life (iBOL) organizes collaborators from more than 150 countries to participate in a variety of
“campaigns” to census diversity among plant and animal groups – including ants, bees, butter ies, sh, birds, mammals, fungi, and
owering plants – and within ecosystems – including the seas, poles, rain forests, kelp forests, and coral reefs. The 10-year Census of
Marine Life, completed in 2010, provided the rst comprehensive list of more than 190,000 marine species and identi ed 6,000
potentially new species.

There is a surprising level of biological diversity, literally in front of our eyes. For example, DNA barcodes showed that a well-known
skipper butter y (Astraptes fulgerator), identi ed in 1775, is actually ten distinct species. DNA barcodes have revolutionized the
classi cation of orchids, a complex and widespread plant family with an estimated 20,000 members. The urban environment is also
unexpectedly diverse; DNA barcodes were used to catalogue 54 species of bees and 24 species of butter ies in community gardens
in New York City.

DNA barcodes are also used to detect food fraud and products taken from conserved species. Working with researchers from
Rockefeller University and the American Museum of Natural History, students from Trinity High School found that 25% of 60 seafood
items purchased in grocery stores and restaurants in New York City were mislabeled as more expensive species. One mislabeled sh

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01/03/2017 DNA Learning Center Barcoding 101
was the endangered species, Acadian red sh. Another group identi ed three protected whale species as the source of sushi sold in DNA barcoding revealed that what
was once thought to be one species
California and Korea. However, using DNA barcodes to identify potential biological contraband among products seized by customs is
of butter y is really ten species with
still in its infancy.
caterpillars that eat di erent plants.

Barcoding relies on short, highly variably regions of the genome. With thousands of copies per cell, mitochondrial and chloroplast
sequences are readily ampli ed by polymerase chain reaction (PCR), even from very small or degraded specimens. A region of the chloroplast gene rbcL – RuBisCo
large subunit – is used for barcoding plants. The most abundant protein on earth, RuBisCo (Ribulose-1,5-bisphosphate carboxylase oxygenase) catalyzes the rst step
of carbon xation. A region of the mitochondrial gene COI (cytochrome c oxidase subunit I) is used for barcoding animals. Cytochrome c oxidase is involved in the
electron transport phase of respiration. Thus, the genes used for barcoding are involved in the key reactions of life: storing energy in carbohydrates and releasing it to
form ATP. COI in fungi is di cult to amplify, insu ciently variable, and some fungal groups lack mitochondria. Instead, the nuclear internal transcribed spacer (ITS), a
variable region that surrounds the 5.8s ribosomal RNA gene, is targeted. Like organelle genes, there are many copies of ITS per genome, and the variability in fungi
allows for their identi cation.

This laboratory uses DNA barcoding to identify plants, fungi, or animals – or products made from them. First, a sample of tissue is collected, preserving the specimen
whenever possible and noting its geographical location and local environment. A small leaf disc, a whole insect, or samples of muscle are suitable sources. DNA is
extracted from the tissue sample, and the barcode portion of the rbcL, COI and ITS gene is ampli ed by PCR. The ampli ed sequence (amplicon) is submitted for
sequencing in one or both directions.

The sequencing results are then used to search a DNA database. A close match quickly identi es a species that is already represented in the database. However, some
barcodes will be entirely new, and identi cation may rely on placing the unknown species in a phylogenetic tree with near relatives. Novel DNA barcodes can be
submitted to GenBank® ([Link] ([Link]

FURTHER READING

Hebert P.D., Cywinska A., Ball S.L., deWaard J.R. (2003). Biological identi cations through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences 270(1512): 313-21.
Hebert P.D.N., Penton E.H., Burns J.M., Janzen D.H., Hallwachs W. (2004). Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butter y Astraptes
fulgerator. Proc Natl Acad Sci USA. 101(41):14812-7.
Hollingsworth P.M. et al (2009). A DNA barcode for land plants. Proc Natl Acad Sci USA 106(31): 12794-7.
Ratnasingham, S., Hebert, P.D.N (2007). BOLD: The Barcode of Life Data System. Molecular Ecology Notes 7(3): 355-64.
Stoeckle M. (2003). Taxonomy, DNA, and the Bar Code of Life. BioScience 53(9): 2-3.
Van Den Berg C., Higgins W.E., Dressler R.L., Whitten W.M., Soto-Arenas M.A., Chase M.W. (2009) A phylogenetic study of Laeliinae (Orchidaceae) based on combined nuclear and
plastid DNA sequences. Annals of Botany 104(3): 417-30.
Benson D.A., Cavanaugh M., Clark K., Karsch-Mizrachi I, Lipman D.J., Ostell J., Sayers E.W. (2013). Nucleic Acids Res. GenBank. 41(D1): D36–D42.

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