DNA Barcoding II: Perfect Open-Ended Experiments for Students
By David Micklos
DNA Learning Center (DNALC), Cold Spring Harbor Laboratory
In a previous article, I offered an introduction to the new science of DNA barcoding and the promise it holds for identifying and preserving the diversity of life on earth. In this article, I’ll explain how you and your students can easily become involved in this exciting field. The upcoming Carolina kit, Using DNA Barcodes to Identify and Classify Living Things, makes it possible to develop DNA barcodes for a variety of plants or animals—or products made from them.
A barcode project
Begin a barcode project by having students brainstorm about a topic that interests them, such as (1) checking for pests or invasive species, (2) monitoring animal movements or migrations, (3) identifying exotic or endangered food products, or (4) detecting food or product fraud. Use a smartphone or digital camera to photograph specimens in their natural environments or where they were purchased or obtained. GPS-enabled devices will tag photos with latitude and longitude, allowing collection sites to be readily placed on a map.
One common application is to inventory biodiversity in an ecosystem, park, or garden. This introduces the concept of a sampling unit, such as the quadrat, a 1-m square area from which each different plant and animal is collected for barcoding. Collected specimens are sorted by visible types or keyed to the family, genus, or species level; then 1 or more representatives of each type or taxa are barcoded. Students may also consider allying with other classes or schools in a “campaign” to systematically inventory a river or ecosystem.
Specimens can include a plant leaf, petal, or bud; an entire insect or insect part; several hair roots (follicles); or flesh from the base of a feather. Fresh, frozen, dried, and even processed food items are also good sources of DNA. DNA can be safely isolated from a small sample of specimen tissue in about 75 min. The sample is ground with nuclei lysis solution, and then proteins and other cellular debris are removed. DNA is precipitated with alcohol, pelleted by centrifugation, dried, and rehydrated.
The purified DNA is mixed with a primer set for either the COI or rbcL barcode region and amplified by polymerase chain reaction (PCR). The animal COI barcode is obtained from the mitochondrial genome, and the plant rbcL barcode from the chloroplast genome. Each cell has 10s to 100s of these organelles with multiple copies of their genomes, so the COI and rbcL barcode sequences are readily amplified—even from very small or degraded specimens. A sample of the amplified DNA is electrophoresed on an agarose gel to confirm a product of about 700 nucleotides for COI and about 600 nucleotides for rbcL.
Sequencing and analysis
The remainder of the amplified DNA is submitted for low-cost sequencing of the barcode region by GENEWIZ® DNA services. This company has optimized reaction conditions for the Carolina kit and produces an excellent-quality sequence with rapid turnaround—usually within 48 hr of sample receipt.
GENEWIZ® sequences are automatically uploaded to the DNALC Web site DNA Subway, an intuitive bioinformatics workflow for analyzing DNA barcodes. At the first stop, students view electropherograms of their barcode sequences, trim the ends, and make a consensus sequence (if both strands have been sequenced). At the second stop, they submit their sequences to the BLAST® Web site to identify close matches in GenBank® and other major databases. Sequences from the BLAST® search and reference data are then added to the analysis. At the final stop, the MUSCLE algorithm aligns the sequences, and phylogenetic trees are generated using the neighbor-joining and maximum likelihood methods. Novel DNA barcodes can be submitted to the database at the Barcode of Life Data System (BOLD).
The Urban Barcode Project
The Urban Barcode Project, funded by a grant from the Alfred P. Sloan Foundation, provides a scalable infrastructure to broadly disseminate DNA barcoding in education. Student research teams at New York City schools develop ideas and submit proposals for barcode projects. Sponsoring teachers receive training and are supported by DNALC staff and scientific mentors at research institutions in New York City. Each team is provided everything needed to develop up to 100 barcode sequences; they may borrow an equipment footlocker to use at their school or attend Open Lab Days at locations around New York City. Barcode teams will present their projects at a symposium in spring 2012, competing for a $10,000 grand prize and $10,000 in runners-up prizes.
A Web microsite (www.urbanbarcodeproject.org) supports all phases of the project. The science of DNA barcoding and suggestions for student experiments are presented in video interviews with scientists and students, animations, an active news feed, and links to The Barcode Blog and iBOL. An online “lab notebook” includes interactive and print versions of the barcode protocol. A Google Maps™ mapping service utility tracks student teams and the specimens they collect.
Together, the Carolina kit and the DNA Subway and Urban Barcode Project Web sites provide the infrastructure for students across the United States to participate in the new science of DNA barcoding.