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Taxonomy

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Introduction

Taxonomy refers to the science involved in the definition of groups of organisms on the

grounds of their common traits. Taxonomical designation are reliant on DNA (deoxyribonucleic

acid) sequence similarities. Given that DNA normally mutates at some known rates, the more

closely related the sequence of DNA of some two organisms, the more lately they diverged from

the shared ancestors. Biologists in consideration of the data on the pair of species are in a

position of constructing the evolutionary tree diagram showing how the existing organisms relate

to some other organisms.

History of taxonomy

The history of taxonomy span over the course of more than 2,000 years. Some millenniums

ago, people used to designate animals, fungi, and plants by whether they were safe to consume,

tasty, poisonous, foul tasting or of medicinal use. One of the earliest known taxonomists was

Aristotle, the Greek Philosopher (384-322 B.C). He organized 500 types of animals as per their

body forms and habitats. Aristotle’s designations were fairly subjective. He believed that animals

that had lungs and gave birth to their live offspring were at the top of living perfection. He also

used polar opposites or dichotomies as a basis for his division of the life’s complexity. In so

doing he divided the organisms into plants and animal. Using dichotomy, he divided the animals

into those with blood and the other without blood in their bodies. Today, the divisions between

the invertebrates and vertebrates correspond to such a classification.

By the 16th century, scientists had discovered countless species that the classification of

Aristotle could have envisioned. Newer schemed persisted to be founded on what was

observable, but the traits considered were normally more mystic than scientific. Scientist such as
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John Ray, Carolus Linnaeus, and George Cuvier continued to classify and contribute to the

taxonomical classification that prevails today. George Cuvier, in particular, contributed to the

broad levels of classifications that include family, class, order, kingdom, division or phylum.
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Figure 1 below shows the present taxonomical scheme.

Figure 1:
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While the current approach to taxonomy as seen in the figure above, is more diverse

that Aristotle’s classification, it is usually lumped into two major taxonomies. They

include Phenetic and cladistics/phylogenic.

Phenetic

Phenetic taxonomy refers to the classification of organisms as per their overall

similarity, normally morphology or any other noticeable characteristics, regardless of

their evolutionary or phylogenic relation. The Phenetic techniques comprise of numerous

ordination and clustering forms. These techniques are refined ways of decreasing the

difference manifested by the organisms to a level that can be considered manageable.

This in practice means measuring of countless variables and presenting them later as

three or two-dimensional charts/graphs. Today, much of the challenges in Phenetic

taxonomy revolves around harmonizing the information loss in the stated reduction

against the simplicity of deducing the resultant graph.

Phonetics does not place emphasis on a particular trait over the rest. Rather, all the

shared traits are equally treated. A computer program then takes this data and process it

through the numeric algorithm to come up with the similarity coefficient, which is a

mathematical extent of similarity, where zero shows no similarity at all and 1 showing

high similarity.

Cladistics/phylogenetic

Phylogenetic taxonomy was invented in the 1950s by Willi Hennig. In this system of

classification, organisms are normally categorized based on the very order they evolved

from shared ancestor. The fundamental idea is that organisms with a shared ancestors

ought to be categorized together, and those with latest shared ancestors are even more
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closely related. Consequently, if an individual desires to investigate a subgroup of some

insects, then features common in insects such as segmented bodies and six legs are

useless. One should instead use a combination of features or individual features that such

a subgroup possess, such as hardened wing for the beetles or butterflies and moths’ scaly

wings. A person, therefore, builds the phylogenies in such a case by attempting to locate

the family tree involving the least feature changes and hence the least amount of

evolution convergent.

DNA barcoding

DNA barcoding refers to the taxonomical techniques that make use of the shorter

genetic markers present in the DNA of an organism to investigate whether it belongs to a

given species. For animals, the most commonly employed barcode region is the segment

of around 600 base pairs of a mitochondrial gene COI (Cytochrome oxidase I). DNA

barcoding was first introduced to the scientific community’s attention by Paul Hebert’s

researchers in 2003. In a paper submitted at the University of Guelph, the researchers

suggested a system through which species could be discovered and identified utilizing the

short section of some DNA from the genome’s standardized region (TRIVEDI, 2016).

Such a DNA sequence could be employed in the identification of some different species,

in the very same manner, a scanner from a convenient store utilizes the black stripes of a

UPC barcode in identifying the individual purchases.

DNA Barcoding procedure

Sampling
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Successful DNA barcoding is reliant on the biological material’s quality. Sampling

protocols are in a position of ensuring that there is sufficient biological samples’

preservation for the study of DNA.

For large invertebrates, birds, fish and mammals

i. The full individual specimen should be placed in a plastic bag and frozen. A write

on the label should be employed in labeling records expedition/vessel, locality, latitude,

name/code, the name of the species, name of the collector and date.

ii. For the bigger specimen with a size that makes it unrealistic to take back to the

laboratory, a small muscle tissue should be taken from any region of the body and frozen

in the cryovial or labeled clip-top grip bag. The vial or the bag should be labeled with the

species name, the scientist involved, locality, vessel code/name and date. Before

discarding, the entire specimens should be photographed, and the digital image cross-

referenced to the code of a tissue sample.

iii. Formalin work areas should be avoided for the handling of specimens.

For invertebrates and small fish

i. For preservation and fixations, 96 percent pure ethanol should be used.

Denaturized alcohol should be avoided at all costs.

ii. The samples should be labeled with the collector’s name, date, coordinated and

locality.

iii. For samples such as kick samples that contain a large amount of water, the

sample liquid should be exchanged with fresh ethanol a day later if practical.

iv. The sample containers should always be filled with ethanol to avert material

damage in the course of transportation. The specimen collection data should be recorded
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on the waxy paper label with the help of a pencil. The ethanol filled jar should be labeled

with the scientist’s name/code, station number, specimen name, date, and locality.

v. Samples should be kept in the dark and cool place to avoid the cases of DNA

degeneration.

Whenever ethanol is unavailable or impractical to obtain in huge amounts, the

specimens ought to be sub-sampled in ethanol. The process of cross-reference labeling

using the distinctive identifiers is vital in lining the tissue samples or sub-samples with

the principal samples. A solution of formaldehyde is capable of degrading the DNA. As

such, if the samples must be fixed in it, it is vital to ensure that they are in a cool place

and transferred to ethanol quickly (within 2 weeks). It is also vital to ensure that

exchange of ethanol takes place after a couple of days.

Specimens for the morphological studies that should be in a dry place like butterflies

should be frozen at -20 degrees Celsius or lower. Alternatively, the sample should be

dried quickly in the incubator or oven.

For plants.

i. DNA of high quality should be acquired from the plant easily once the tissues are

rapidly dried. A majority of botanist routinely make use of silica gel for the leaf material

field preservation.

ii. The silica gel to leaf tissue ratio should be about 5-10:1. The tissues must be dry

for the best results.

iii. The healthy and green leaves from a given plant should be chosen. A minimum

of about 2 to 3 square centimeter is generally accepted for 1 to 2 DNA extraction, though

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some small individuals can have considerably less leafy material. More is however

preferred as a rule of thumb.

iv. It is important to track the individual from which the leaf material is sampled.

Tiny tags are effective and do not get in the way of some frequent pressing activities.

Preservation of voucher specimens is vital particularly from individuals with association

with the material acquired for DNA studies. The sample of DNA without the associated

voucher specimen have very little value in research.

v. The leaf tissue should be placed into the small sealable container with silica gel.

The leaf materials should be cut into tiny pieces prior to inserting them into the sealable

container to increase the leaf’s surface are that is placed in silica. This way, the process of

drying is swift.

vi. The individual’s collection number should be written down on the small paper

piece and inserted into a Ziploc bag. The same information should be written on the bag’s

exterior with the permanent marker.

vii. The silica gel packets should be stored in the sealed container or bag to ensure

that moisture is kept out while at the same time avoiding the risks of re-hydration over

the course of time. Packets of silica gel should be stored in the freezer for the long-term

storage.
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Extraction of DNA

Total cellular plant DNA isolation using the CTAB procedure

i. 10 ml of the isolation buffer with 80 80 ml beta-mercaptoethanol is preheated in a

water bath at 65 degree Celsius. The pestle and mortar should be heated.
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ii. 0.5 g to 1.5 g of leaf tissues (fresh) should be ground using the preheated mortar

and pestle. Some of the isolation buffer should come in handy in this process. Silica gel

or sterile sand could be added to grind the tougher tissues. The remaining buffers should

be added and swirled to append the slurry (Powel nd: 13). The slurry should then be

poured into the 50 ml tube and incubated for about 20 minutes at a temperature of 65

degrees Celsius with the optional gentle swirling from time to time.

iii. 10 ml of SEVAG mixture should be added, mixed thoroughly but gently. The

tube’s cap should be opened to release the gas. It should then be re-tightened and the tube

rocked utilizing the orbital shaker for 1 hour at a speed of 100 to 150 rpm.
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iv. 20 minutes should be enough at a temperature of 25 degrees Celsius and a spin of

4000 rpm. The aqueous upper phase with the DNA becomes colorless and clear.

v. The aqueous upper phase with the DNA should be transported to the clean tube

using the plastic transfer pipette. The plant debris and SEVAG should be disposed in the

designated waste container.

vi. The aqueous phase’s volume should be estimated (usually 15 ml) and the two

third volume of iso propanol (- 20 degree Celsius) added and mixed gently, It should be

placed in a freezer at -20 degrees Celsius for a couple of hours for the fresh samples or 14

days for the silica dried samples to precipitate the DNA.

vii. A centrifuge should be used to spin the sample for 5 minutes at a speed of 3000

rpm to collect the precipitate. The liquid should be poured off after spinning and added

about 3 ml of the 70 percent ethanol. Pellets should be dislodged to facilitate the process

of washing for about 5 to 6 minutes. This step should be repeated twice.

viii. The DNA should be spun down for five minutes at a speed of 3000 rpm. The

liquid should be poured off and the apparatus drained in an inverted manner to permit the

evaporation of the alcohol.

ix. DNA should be re-suspended in a TE buffer or 1 to 15 ml H20. The sample

should be transferred into the eppendorf tube (1.5 ml) and stored at - 80 degrees Celsius

for a long period or – 20 degrees Celsius for a short time.

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Purification of DNA from the Animal Tissue (Qiagen Spin-column procedure)

i. 25mg tissue should be cut into some small pieces, and placed in the 1.5 ml

eppendorf tube. 180 ml Buffer ATL should then be added and the tube marked

appropriately.

ii. 20 ml proteinase K should be added and thoroughly mixed by vortexing. It should

then be incubated at a temperature of 56 degrees Celsius till the tissue is lysed

completely. Vortexing should be done occasionally in the course of incubation to diffuse

the sample. The time of lysis varies mostly on the tissue type processed. The usually time

is however 1 to 3 hours.

iii. After vortexing for about 25b second, Buffer AL should be added to the sample

and thoroughly mixed by vortexing. 20 ml of 96 to 100 percent ethanol should be added

and thoroughly mixed again through vortexing.

iv. The mixture from the above step should be pipetted into a spin column located in

the collection tube (usually 2ml). After centrifuging at about 8000 rpm for 60 seconds,

the flow-through should be discarded.

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v. 500 ml buffer AW1 should be added into a spin column with care taken not to

destroy the filter. After a minute of centrifuging at a speed of 800rpm the flow-through

should be discarded.

vi. 500 ml Buffer AW2 should be added into a spin column. Care should also be

taken to avert filter damage. After a three minutes centrifuge at a speed of 13000rpm, the

flow-through should be discarded.

vii. Another one minute should be spent in centrifuge again to ensure the dryness of

the spin column membrane.

viii. The spin column should be placed in the clean eppendorf tube (1.5ml). 150 ml

Buffer AE should be pipetted directly onto the membrane of the spin column (DNeasy

spin column). Care should be taken to make sure that the membrane is not damaged. 15

minutes should be enough to incubate at room temperature and centrifuged at a speed of

8000 rpm for 2 minutes to elute.

ix. Samples should be stored at -80 degrees Celsius for extend period or – 20 degrees

Celsius for a short period of time.

Silica-column purification of DNA (Using QIAquick)

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i. Labeled silica-columns should be inserted into the vacuum manifold.

ii. 750 ml Buffer PB (5 volumes) should be added to a single volume of the total

DNA (150 l). Samples should be loaded into silica-columns and vacuum applied till

the whole sample passes through the column.

iii. 750 ml Buffer PE should be added to each of the column and vacuum applied.

The main goal of this step is to wash the content.

iv. Vacuum should be switched off and columns transferred to the

availed collection tubes (2ml preferably). To get rid of the residual ethanol the centrifugal

process should be done for 60 second at a speed of 13000rpm.

v. The sterilized eppendorf tube’s lids should be cut and the columns

placed into such tubes. 50 ml buffer EB should be added (or some sterilized water) onto
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the column’s membrane and left for 60 seconds at room temperature to ensure that the

DNA dissolve in an elution buffer.

vi. 1 minute should be spent spinning at a speed of 13000rpm. Caps

should be replaced onto tubes and DNA levels verified on the agarose gel.

DNA level verification on the 1 percent agarose gel

i. The sides of the gel tray should be taped to maintain a hold of the gel in the

course of setting. It should then be placed well to form combs into a tray.

ii. 1 percent agarose gel should be prepared through mixing about 1.5 gram with the

TBE buffer. The mixture should be boiled in a microwave up to the point where the

agarose is completely dissolved (it might take about two minutes). It should be cooled by

running some cold water. Once it is cool 6 ml Ethidium Bromide should be added and

swirled to mix. The gel should be poured and left for at least thirty minutes prior to

loading the samples. Combs should be removed and the gel placed into the

electrophoresis tank.

iii. 5 ml of the loading Buffer should be spotted for each of the sample of DNA on

the parafilm strip.

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iv. 5 ml of the DNA should be mixed with a loading buffer using a pipette. The mix

should be loaded into the well on a gel. It is advisable to run it for about half an hour at

approximately 110 Milli Amps.

v. The gel should be observed under an ultraviolet light box, as well as a

photographic gel. The quantity of DNA added to a mix of PCR depends on the total DNA

concentration.

Polymerase chain reaction

a. A PCR experiment sheet should be filled out and appropriate numbers of the

PCR tubes labelled. It is prudent to prepare both a negative and positive control. The

positive control entails the known sample previously worked on for an amplified region.

On the other hand, the negative entails the mixture of PCR without the addition of DNA

to look for contagion.

b. A master mix should made comprising of all ingredient other than the template

DNA in the eppendorf tube, thereby allowing for another sample. For instance, for 6
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samples one should make sufficient master for about 7. Adding the ingredients in the

following manner is recommended.

i. PRC master mix (greenish top for the nuclear regions and reddish top for the

plastids each reaction with 22.5 l).

ii. BSA (0.8 ml for each reaction).

iii. Reverse (0.3 ml for each reaction).

iv. DMSO (0.5 ml for the nuclear region only).

c. The mix should be vortexed and briefly centrifuged where necessary, like in the

case of the mix being on the top or side of the tube.

d. A master mix amounting to 23.9 ml should be divided into some individual

reaction test tubes. Template DNA should be added to each of these individual reaction

tube. However, no DNA should be added to the set negative control. Where it is added

the amount depends on the DNA’s strength from the agarose gel.

e. The individual mixes should be briefly spin down to make sure that no solutions

remain on the tube’s sides.

f. The tubes should be loaded into a PCR machine and necessary program for a

given area amplified.

g. The PCR product levels should be checked by running them on the agarose gel.

Quantifying and cleaning PCR products

a. 5 Buffer PB volumes (of 100 ml) should be added to a single volume of a 20 ml

PCR reaction and mixed.

b. Silica-columns should be prepared and placed on the vacuum manifold.

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c. Samples should be binded into columns and vacuum applied to bind the DNA.

The vacuum should be switched off once the samples are through the columns.

d. 0.75 ml Buffer PE should be added to each of the column and vacuum applied to

each.

e. Each column should be transferred to the collection tube (0.2 ml) and centrifuged

for 60 seconds.

f. Each column should be placed into the clean and lidless eppendorf tube (1.5ml).

g. 20 ml Elution Buffer should be added directly onto a membrane to elute the DNA.

Alternatively, H20 should be used. Incubation should follow for 20 minutes at room

temperature and centrifuged at 1300rpm for 2 minutes.

h. A spectrophometer reading ought to be read to establish the concentration of the

DNA.

i. After the purification of the DNA, it should be subjected to sequencing. The

sequence should in turn be submitted to some global databases such as the NCBI tasked

with the provision of accession number subsequent to a successful incorporation.

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Bibliography

Powel, M., Michele Van der Bank, Maurin, O. and Savolainen, V. (n.d). DNA

Barcoding: A practical guide. Canadian Museum of Nature.

TRIVEDI, S., ANSARI, A. A., GHOSH, S. K., & REHMAN, H. (2016). DNA

Barcoding in Marine Perspectives: Assessment and Conservation of Biodiversity. Cham,

Springer International Publishing.

PELLETIER, B. (2016). EMPIRE BIOTA: taxonomy and evolution 2nd edition. [S.l.],

LULU COM.