Foundations In Biology: Ecology & Evolution LAB

LAB 4

LABORATORY 4: DNA Barcoding: DNA Extraction & PCR

Objectives of this laboratory:
1. To become familiar with standard molecular laboratory procedures.
2. To learn about DNA structure through the extraction process.
3. To use a search engine to find a scientific journal article about a particular
topic.
4. To collect and analyze sequence data from plants, and use DNA to identify
these species.
Introduction to DNA Barcoding
Taxonomy, the science of classifying living things according to shared features, has
always been a part of human society. Carl Linneas formalized biological classification
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
effects 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
animalslet alone microbesliving in many unique ecosystems on earth. Less than two
million of the estimated 550 million plant and animal species have been identified.
Scientists agree that the yearly rate of extinction has increased from about one species per
million to 1001,000 species per million. This means that thousands of plants and
animals are lost each year. Most of these have not yet been identified.
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. Differentiating subtle anatomical differences between closely
related species requires the subjective judgment of a highly trained specialistand few
are being produced in colleges today.
Now, DNA barcodes allow non-experts to objectively identify specieseven from small,
damaged, or industrially processed material. Just as the unique pattern of bars in a
universal product code (UPC) identifies each consumer product, a DNA barcode is a
unique pattern of DNA sequence that identifies each living thing. Short DNA barcodes,
about 700 nucleotides in length, can be quickly processed from thou- sands 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,
fungi, and animal groupsincluding ants, bees, butterflies, fish, birds, mammals,
mushrooms, and flowering plantsand within ecosystemsincluding the seas, poles,
rain forests, kelp forests, and coral reefs. The 10-year Census of Marine Life, completed
in 2010, provided the first comprehensive list of more than 190,000 marine species and
identified 6,000 potentially new species.

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There is a surprising level of biological diversity, literally in front of our eyes. For
example, DNA barcodes showed that a well-known skipper butterfly (Astraptes
fulgerator), identified in 1775, is actually ten distinct species. DNA barcodes have
revolutionized the classification 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 butterflies in
community gardens in New York City.
DNA barcodes are also used to detect food fraud and products taken from con- served
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 fish was the endangered species,
Acadian redfish. Another group identified three protected whale species as the source of
sushi sold in California and Korea. However, using DNA barcodes to identify potential
biological contraband among products seized by customs is still in its infancy.
Barcoding relies on short, highly variably regions of the genome. With thousands of
copies per cell, mitochondrial and chloroplast sequences are readily amplified by
polymerase chain reaction (PCR), even from very small or degraded specimens. A region
of the chloroplast gene rbcLRuBisCo large subunitis used for barcoding plants. The
most abundant protein on earth, RuBisCo (Ribulose-1,5- bisphosphate carboxylase
oxygenase) catalyzes the first step of carbon fixation. 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 difficult to amplify, insufficiently 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 identification.
This laboratory uses DNA barcoding to identify plants. First, a sample of tissue is
collected, preserving the specimen whenever possible and noting its geographical
location and local environment. DNA is extracted from a small sample of your plant, and
the barcode portion of the rbcL gene is amplified by PCR. The amplified 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
identifies a species that is already represented in the database. However, some barcodes
will be entirely new, and identification may rely on placing the unknown species in a
phylogenetic tree with near relatives. Novel DNA barcodes can be submitted to
GenBank (www.ncbi.nlm.nih.gov).

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LAB 4

PART 1: Collect, document and identify your specimen.

Procedure:
1. Collect specimens according to a strategy outlined by your instructor.
2. Use a smartphone or digital camera to photograph your specimen in its natural
environment.
a. Take wide, medium, and close-up views.
b. Include a person for scale in wide and medium shots. Include a ruler or coin for
scale in close-ups.
3. Use a field guide or taxonomic key to identify your specimen as precisely as possible: kingdom > phylum > class > order > family > genus > species. Taxonomic keys
for local plants, fungi, or animals are often available online, at libraries, or from
universities, natural history museums, and botanical gardens.
PART 2: DNA Extraction
The first step in acquiring molecular sequences is to extract the DNA from the cells of an
organism. Genetic material is typically found enclosed within the nucleus or organelles
of cells, along with many other types of molecules, so it must be extracted from the cells
before it can be further analyzed.
The extraction of DNA involves three distinct steps:
1. Cell Lysis: The DNA is enclosed within the cell and nuclear
membranes, so the first step in DNA extraction is to get the DNA out of
the cells. Cells will be lysed (broken open) to rupture the cell walls and
cell membranes. First the sample will be macerated; then we will use a
buffer (AP1) to break up the lipids surrounding the cellular membranes.
This basically breaks open cell and nuclear membrains. The dilemma here
is that it also exposes DNA to proteins in the plant tissue. Therefore, the
enzyme Proteinase K must be added to denature the proteins and keep the
DNA intact.
2. Elimination of Cellular Debris: Once you have destroyed the
hydrolytic enzymes you can begin the DNA purification process. In
essence you will put the cellular components into a spin column and
remove the DNA. The DNA sticks to a membrane across the column, the
proteins and other cytoplasmic components pass through. This is
accomplished by adding a protein precipitating solution and buffers
(Buffer AL) as well as ethanol. Upon centrifugation, the material will pass
through the membrane that will attract the DNA and allow debris to pass
through. Two wash steps follow this with the addition of buffers AW1 and
AW2.
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3. DNA Elution: 3. You will now remove the DNA from the matrix by
adding an elution (removal) buffer, Buffer AE. Spinning the tube with the
DNA embedded in the matrix will pull the elution buffer through the
matrix, thus eluting the DNA.
Procedure:
1. Take a small piece of your specimen (no larger than a inch square) and put it in a
1.5ml tube. Then grind up this material.
2. Add 400 L Buffer AP1 and 4 L RNase A. Vortex and incubate for 10 minutes at
65 C. Invert the tube 2-3 times during incubation.
3. Add 130 L Buffer P3. Mix and incubate on ice for 5 minutes.
4. Centrifuge the lysate (your solution) for 5 minutes at 13,300 rpm.
5. Pipet the lysate into a QIShredder spin column with collection tube. Centrifuge for 2
minutes at 13,300 rpm.
6. Transfer the flow-through into a new tube without disturbing the pellet if present.
Add 1.5 volumes of Buffer AW1, and mix by pipetting.
7. Transfer 650 L of the mixture into a DNeasy Mini spin column placed in a
collection tube. Centrifuge for 1 minute at 8,000 rpm. Discard the flow-through.
Then, repeat this step.
8. Place the spin column into a new collection tube. Add 500 L Buffer AW2, and
centrifuge for 1 min at 8,000 rpm. Discard the flow through.
9. Add another 500 L Buffer AW2. Centrifuge for 2 minutes at 13,300 rpm.
NOTE: Remove the spin column from the collection tube carefully so that the column does not come
into contact with the flow-through.

10. Transfer the spin column to a new 1.5 ml tube, then add 100 L Buffer AE for
elution. Incubate for 5 minutes at room temperature. Centrifuge for 1 minute at 8,000
rpm. This is your extracted DNA label your tube clearly!
PART 3: Barcode amplification (PCR)
Now that we have isolated our DNA, the portion of DNA of interest (rbcl) must be
amplified (in other words, lots of copies of it are made) using a procedure called PCR
(Polymerase Chain Reaction). PCR (short for Polymerase Chain Reaction) is a
relatively simple and inexpensive tool that you can use to focus in on a segment of DNA
and copy it billions of times over.
The PCR reaction mixture contains:
an extracted sample of double-stranded DNA from a biological sample, to act as a
template,
two short, artificially synthesized primers that are complementary to the ends of
the sequence to be amplified,
the four dNTPs (deoxyribonucleoside triphosphates), which are the substrate for
DNA replication (the building blocks of the DNA copies)
a DNA polymerase (an enzyme required to copy DNA) that can tolerate high
temperatures without being degraded, and
salts and a buffer to maintain a near-neutral pH.
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The PCR amplification is a cyclic process in which a sequence of steps is repeated over
and over again:
The first step involves heating the reaction to near boiling point, to separate
(denature) the two strands of the DNA template.
The reaction is then cooled to allow the primers to bind (or anneal) to the
template strands.
Next, the reaction is warmed to an optimum temperature for the DNA polymerase
to catalyze the production of complementary new strands (extension = make
copies).
A single cycle takes a few minutes to produce two copies of the target DNA sequence,
leaving the new DNA in the double-stranded state. Repeating the cycle many times leads
to an exponential increase in the number of copies of the DNA sequence. In the early
days of PCR, these cycles were carried about by repeatedly moving samples among water
baths held at the different temperatures, an extremely tedious procedure! Now, PCR
machines automate the entire process and quickly and reliably cycle the samples through
the appropriate temperatures.

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Procedure:
1. Obtain PCR tube containing Ready-To-Go PCR Bead. Label the tube with your
identification number
2. Use a micropipette with a fresh tip to add 23 L of the primer/loading dye mix to
each tube. Allow the beads to dissolve for 1 minute.
3. Use a micropipette with fresh tip to add 2 L of your DNA (from Part II) directly into
the appropriate primer/loading dye mix. Ensure that no DNA remains in the tip after
pipetting.
4. Store your sample on ice until your class is ready to begin thermal cycling.
5. Place your PCR tube, along with those of the other students, in a thermal cycler that
has been programmed with the appropriate PCR protocol.
Your PCR will be amplified using this protocol:
Initial step: 94C 1 minute
35 cycles of the following profile:
Denaturing step: 94C
Annealing step: 54C
Extending step: 72C

15 seconds
15 seconds
30 seconds

Then, one final step to preserve the sample: 4C ad infinitum

PART 4: Gel Electrophoresis

A convenient way to separate or purify DNA fragments is by gel electrophoresis.
Samples containing the fragments are placed in wells at one end of a semisolid gel and an
electric field is applied to the gel. Because of its phosphate groups, DNA is negatively
charged at neutral pH; therefore, because opposite charges attract, the DNA fragments
move through the gel toward the positive end of the field. Because the spaces between
the polymers of the gel are small, small DNA molecules can move through the gel faster
than larger ones. Thus, DNA fragments of different sizes separate from one another and
can be detected with a dye. This gives us three types of information:
The number of fragments. Gel electrophoresis can provide some information
about the number of times a specific DNA sequence occurs in a DNA sample.
The size of fragments. DNA fragments of known size are often placed in one
well of a gel to provide a standard for comparison. This tells us how large the
DNA fragments in the other wells are.
The relative abundance of a fragment. In many experiments, the investigator is
interested in how much DNA is present. The relative intensity of a band
produced by a specific fragment can indicate the amount of that fragment.
After separation on a gel, a fragment with a specific DNA sequence can be revealed with
a single-stranded DNA probe. This fragment could then be analyzed in terms of
sequence or amplified and used experimentally.

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In this lab, we are using gel electrophoresis to see if your PCR amplified a region of
DNA that is the approximate size of the rbcl gene (between 550 600 nucleotides) that
we are trying to amplify.

This lab is modified from:

DNA Learning Center. 2014. Using DNA Barcodes to Identify and Classify Living Things. Cold
Springs Harbor Laboratory.
2014. DNA Extraction Lab. Discover the Microbes Within: The Wolbachia Project. Cornell
University.

Foundations In Biology: Ecology & Evolution LAB

LAB 4

Lab 4 Worksheet
General Questions
1. What is a DNA barcode?

2. If we were using animal tissue instead of plant tissue why would we not use rbcl as
our barcode?

Part 2: Extraction
3. Describe the effect of each of the following steps or reagents used in DNA isolation:
i. Collecting fresh or dried specimens

ii.

Using only a small amount of tissue

iii. Grinding tissue with pestle

iv. Lysis solution

v.

Heating of lysate

Part 3: Replication
4. Briefly explain how the PCR process takes advantage of how DNA behaves at
different temperatures.

Foundations In Biology: Ecology & Evolution LAB

LAB 4

5. You used a PCR tube containing Ready-To-Go PCR Bead. These beads contain buffer
salts, nucleotides and Taq polymerase in a lyophilized powder. Describe the purpose of
each component in your own words by filling out the table.
Component
Function
Taq polymerase

Buffer salts

Nucleotides

Part 4: Gel Electrophoresis

6. Looking across your gel at the PCR products, do the bands all appear to be the same
bp size and intensity?

7. Which samples amplified well, and which ones did not? Give several reasons why
some samples may not have amplified; some of these may be errors in procedure.

8. Briefly explain how the gel electrophoresis process takes advantage of the charge of
DNA molecules to determine the size of DNA fragments.

9. If we used COI as a barcode for animal tissue and then ran the PCR products on a gel,
would you expect the products to be the same lengths as our products (for rbcl)?
Explain your answer.