Introduction to DNA Barcoding

copy/amplify COI gene fragment with PCR
To accelerate species discovery and better protect biodiversity,
scientists working under the International Barcode of Life (iBOL) project
are building a digital genetic registry of Earth's eukaryotic life
using a DNA barcode system
iBOL scientists are storing this information in the
Barcode of Life Data Systems (BOLD)
BOLD assigns two digital pages to each
biological specimen in the database
A specimen page contains information about an organism and where it was collected
http://ibol.org
The reference barcode records in BOLD can be used to identify unknown specimens
Together, the specimen and sequence pages for a given plant or animal constitute a REFERENCE DNA BARCODE RECORD
A sequence page contains the COI gene sequence (DNA barcode) obtained from the same organism
BOLD currently contains about 2 million reference barcode records from
animals, plants, fungi and other life
For fisheries research, it is important to know the species names of fish embryos that are collected in seawater samples
However, a query DNA barcode sequence for this embryo can be generated in the lab within a short amount of time
The query barcode sequence can then be used
as a search string in the BOLD Identification System
Because this early life form lacks
adult anatomical features, it cannot be assigned a species name
BOLD compares the DNA search string (query sequence) against
reference barcode records and retrieves the names of species
in the database with similar DNA sequences
The full potential of BOLD as a species recognition tool relies on populating the database with reference barcode records from as many specimens as possible
Scientists around the globe are now conducting campaigns to create reference barcode records for species with the highest socioeconomic important to humanity
Here's a simple example of how it works
This information can help scientists understand the health of important fish stocks and how they are affected by
natural events and human activities
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The rate of species loss from human activities threatens to outpace the rate of species discovery using this classification system
This classification system is slow and relies on a small number experts to formally describe and name new species
Stems, prostrate or climbing, up to 2m long, cylindrical 4-5mm wide with many small rosettes and short shoots at the base, sometimes adventitious underground stems produce rosettes away from the main plant; internodes 20-40mm long. Leaves of the rosettes, coriaceous, semi-amplexicaul, lanceolate 40-50mm long, 10-15 mm wide, obtuse to acuminate at the apex, auriculate at the base clasping the stem for 2/3 the diameter. Pennate veins, irregularly reticulate, running obliquely to the margin; longitudinal veins, 2 or 3 on each side, running in the outer 1/2 of the blade; originating from the midrib near the base of the leaf, converging at the tip. Tendril, slender 1.5mm wide, 2-3 X as long as the leaf, descending without curl. Leaves of the short shoots often larger than those of the rosette and climbing stem, 140mm long, 40mm wide. Leaves of the climbing stem similar to those of the rosette, 70-120mm long, 15-30mm wide; leaves tending to reduce in size as the stem grows longer. Tendrils the same length as the leaves, slender, 1mm wide, straight without curl and bearing no pitchers. Pitchers of the rosettes, abruptly incurved at the front or side from the hanging end of the tendril, 40-55mm tall, 15-20mm wide, ovate-ellipsoidal in the lower 2/5 narrowing slightly about 5mm below the mouth, ellipsoidal at the back and above the narrow part with 2 fringed wings over the entire length. Wings 1-2mm wide, fringe segments filiform, 2-4mm long, and 8-14 fringe segments per cm. Mouth, very oblique, elevated towards the lid, ovate becoming acute towards the lid with almost no neck; peristome, rounded 1.5-2mm wide in front becoming flattened and widest, 2.5-3.5mm in the upper 1/3 with ribs 0.3-0.6mm apart, 6-8 X as long as wide on the inner margin, pectinate. Inner surface of the pitcher, glandular in the lower 2/ 3 with minute elliptic to rotund overarched glands 0.15-0.3mm long, the long axis of the glands orientated at right angles to the long axis of the pitcher; from the bottom to the top 400-600 glands per cm2, glands becoming larger and more rotund towards the bottom of the pitcher; the upper 1/3 glandless and pruinose...
Retail stores and supermarkets often use a UPC (Universal Product Code) barcode system
The UPC barcode contains a unique combination of bars and spaces that identifies each product sold by a company
In order for a barcode to be useful for
a consumer and a company, it must be linked to certain types of product information
Manufacturer: Acme, North America
Volume: 500 mL
Product Category: Health and Beauty
Product Name: Clean and Shine
Price: $3.99
Product Record
When a printed barcode is scanned at the checkout counter, it is digitized and compared against the product records in the company database
When a match is found for the barcode in the database, the product information linked to the barcode is retrieved
Manufacturer: Acme, North America
Volume: 500 mL
Product Category: Health and Beauty
Product Name: Clean and Shine
Price: $3.99
Product Record
For animals, a DNA barcode is a short
segment of a mitochondrial gene
A digital representation of each barcode is linked to product information in a company database
When the COI gene sequence is compared between members of the same species, few nucleotide differences are observed
When the COI gene sequence is compared between members of different species, more nucleotide differences are observed
based on these differences, the nucleotide sequence of the COI gene (i.e. the DNA barcode) can be used to uniquely identify animal species
cytochrome c oxidase
subunit 1
Pacific Sanddab 1
Pacific Sanddab 2
Pacific Sanddab
Speckled Sanddab
Pacific Sanddab 1
Citharichthys sordidus
Pacific Sanddab
Citharichthys sordidus
Speckled Sanddab
Citharichthys stigmaeus
Pacific Sanddab 2
Citharichthys sordidus
In order to be useful as a species identification tool, a DNA barcode must be linked to specific forms of information stored in a database
as of 25 January 2013
extract
genomic DNA
copy/amplify COI gene fragment with PCR
verify size of COI gene fragment by gel electrophoresis
purify and sequence
COI gene fragment
analyze and edit
COI gene sequence
The workflow often begins in the field with the collection of target specimens and tissue samples
Data associated with each specimen and where it was collected is then uploaded to BOLD
to create a specimen page
Reference DNA barcodes are then generated
in the lab using standard techniques
The DNA barcode sequence data is then uploaded to BOLD to create a sequence page that is linked to the specimen page
extract
genomic DNA
verify size of COI gene fragment by gel electrophoresis
purify and sequence
COI gene fragment
analyze and edit
COI gene sequence using
bioinformatics tools
tissue
sample
Benefits of participating in this global biodiversity project
as a citizen scientist
receive authorship for your contribution of genetic data to BOLD
expand your scientific knowledge in preparation for a scientific career
apply cutting-edge scientific tools and technologies for a global scientific project
develop a realistic impression of what scientists do on a day-to-day basis
get a rare glimpse into your scientific career opportunities
gain an appreciation for how scientific disciplines are interconnected
tissue
sample
tissue
sample
tissue
sample
fish specimen
echinoderm specimen
crustacean specimen
specimen page
sequence page
Nepenthes xiphioides
1/605 differences
93/605 differences
pollution
habitat destruction
overharvesting
invasive species
Let's explore the barcoding metaphor
Reference DNA barcode records are
created through a multi-step workflow
In October 2010, the International Barcode of Life project was formally launched with the goal of generating 5M barcode records by October 2015
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eBOL is a new campaign that seeks to engage students in DNA barcoding
a partial list of DNA barcoding campaigns
The eBOL website contains a suite of new resources for students to learn and apply the concepts and methods of DNA barcoding in the classroom
Of Earth's estimated 10 - 50 million species, fewer than 2 million have been named in the last 250 years using Linnaean taxonomy
fishes
marine life
fungi
mammals
ants
polar life
butterflies
and moths
birds
for more information on how your students can get involved in DNA barcoding visit

www.educationandbarcoding.org