BET305/4

INSECTS BIOLOGY AND SYSTEMATICS

LAB REPORT
INSECT MOLECULAR GENETICS

SEMESTER 1 2020/2021

Prepared by:

RAHMAH HAYATI BINTI MOHD FAUZI

School:

SCHOOL OF EDUCATIONAL STUDIES

Prepared for:

ASSOC. PROF. DR. ABDUL HAFIZ AB MAJID

Submission Date:

4th FEBRUARY 2021

Introduction

Insect molecular genetics is the study of molecular structure of DNA in insect that

can be useful in insect pest management, sex determination, insect behaviour,

systematics and ecology. In this study, understanding the routine procedure in the

laboratory is fundamental to get the information of the desired DNA. Method such as

Polymerase chain reaction (PCR), DNA extraction, Gel Electrophoresis and The

Northern blot are commonly used in laboratory. Gel electrophoresis is the technique that

often used in insect molecular genetics. In fact, it contributes in identifying immatures

and sibling species (Avise, 1974). In this practical, we require to practice technique to

lyse the cells gently and solubilise DNA. Besides, removing contaminating protein, RNA

or macromolecules by enzymatic and chemical reaction for soldier termites of

Coptotermes gestroi. The result of DNA fragments extracted is used to determine DNA

band under gel electrophoresis.

Coptotermes gestroi or commonly known as Wasmann has been a threat to the

wooden structure as a damaging termite. They are also recognised as “Asian

subterranean termite”. The infestation of Coptotermes gestroi in a short amount of time

by a large, mature colony has resulted in a very bad condition to the wooden structure

(Scheffrahn, R. H., & Su, N. Y., 2000). This species has been found in many parts of the

world such in Asia, New World tropics (Brazil and Barbados) southern Mexico, the

Southeastern United States, some islands of the West Indies, the Marquesas Islands

(Pacific Ocean), Mauritius and Reunion Islands (Indian Ocean) and recently in Taiwan

(Yeap, B. K., Othman, A. S., & Lee, C. Y., 2011).

Procedures

A) DNA Extraction

Five soldiers of termites of Coptotermes gestroi is washed with 70% ethanol then

rinsed with ddH2O. The head of the termites is cut by using forceps and transferred into

1.5 ml microcentrifuge tube. 200 μl of QGT buffer is added and the sample is

homogenised by sterile micro-pestle. 20 μl of Proteinase K is added and vortexed

thoroughly. The sample is incubated at 60 ° C for 15 minutes. 200 πl of QGB Buffer is

added and shook vigorously for 10 seconds. 200 μl of absolute ethanol is added to

sample lysate and is mixed immediately by shaking vigorously for 10 seconds. The GD

Column is placed in a 2 ml Collection Tube. All mixtures are transferred to the GD

Column. The sample is centrifuged at 14000rpm for 1 minute. 2 ml flow-through in the

Collection Tube is discarded and the GD Column is transferred to a new 2 ml Collection

Tube. 400 μl of W1 Buffer is added to the GD Column. The sample is centrifuged at

14000rpm for 30 seconds then the flow-through is discarded. The GD Column is placed

back into the Collection Tube. 600 μl of Wash Buffer is added to the GD Column. The

sample is centrifuged at 14000rpm for 30 seconds then the flow-through is discarded.

The same Collection Tube is used to place the GD Column.

Next, the sample is centrifuged for 3 minutes at 14000rpm to dry the column matrix.

The following steps are repeated twice. The GD Column that is dried being transferred

to a clean 1.5 ml microcentrifuge tube. 50 μl of pre-heated Elution Buffer is added into

the center of the column matrix. The sample is stood for at least 3 minutes to allow the

Elution Buffer to be completely absorbed. The sample is centrifuged at 14000rpm for 30

seconds to elute the purified the DNA.

 B) Polymerase Chain Reaction

1. The reaction mixture in a PCR tube is prepared by pipetting each reagent in

volumes according to the following table.

Components Amount ( μl ¿

RNase- free water or ddH2O 7

Taq PCR Master mix 12.5

10x Primer F (10 πM ¿ 0.25

10x Primer R (10 μM ) 0.25

Template DNA 5

Total volume 25

2. The mixture is resuspended gently. The PCR tube is placed into a thermal cycler.

The PCR cycling is set according to the following] table.

Step Temperature (° C) Time (min) Numbers of cycles

Pre-denaturation 95.0 5 1

Denaturation 95.0 1

Annealing 55.0 1 35

Extension 72.0 2

Final extension 72.0 10 1

Store 4.0 ∞
 3. After completing the cycles, the PCR tube is removed from the thermal cycler

and is stored at – 20 ° C to proceed in the gel electrophoresis.

C) Gel Electrophoresis

Step 1: Gel Preparation

1% Agarose powder is dissolved in 1x TBE/TAE buffer. The mixtures are heated

until agar dissolved. The liquid is let to slightly cooled down by water bath and mixed

with 5 μl volume of RedSafe nucleic acid stain. The buffer is poured into gel cast and

the gel comb is put on. The buffer is left to solidify in room temperature for 20-30

minutes. The gel comb is removed. The gel cast is transferred to gel tank and is

filled with 1x TBE/TAE buffer.

Step 2: DNA & PCR Product Loading and Gel Electrophoresis

3 μl of DNA loading dye is pipetted onto parafilm to form droplet. 5 μl of DNA

sample (DNA extraction and PCR reaction) is mixed with the DNA loading dye by

pipetting. The DNA mix is pipetted into the consecutive well. 5 μl of thermo Scientific

GeneRuler 1 kb DNA ladder is pipetted into the first well. Gel electrophoresis is

performed at 90V for minutes.

Result

Result obtained by Group 6

Discussion

1. Explain in detail the mechanism of gel electrophoresis and the use of Agarose in

separating DNA fragments.

It is important to understand the basic protocol to separate DNA fragments in

molecular genetics. Agarose has been widely used in DNA separation as it is the most

effective way to separate DNA fragments of varying sizes ranging from 100 bp to 25

kb1. Agarose polymers binds non-covalently and form a network of bundles whose

pores sizes determine a gel’s molecular sieving properties. The sizes of DNA fragments

that will be separated will determine the concentration of the agarose in a gel. Most of

the time, the gels ranging within 0.5%-2%.

As the DNA that mixed with loading dye is loaded into pre-cast well in the gel, the

current is applied. The DNA fragments move to the positive electrode because the

phosphate backbone of the DNA molecule is negatively charged. DNA has a uniform

mass or charged ration thus is easier to separate by size in a pattern that shows the

distance travelled is inversely proportional to the log of its molecular weight. The ladder

that we observed under UV light is the separated DNA that determine the sizes of the

bands. The sizes of DNA fragments can be determined by comparing to the DNA

standard or DNA ladders. (Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H., 2012)
 2. Describe what you observed on the gel (include details such as smear, bands at

what size etc.) and explain your observation or reasons to the observation.

From the gel electrophoresis, our group (Group 6) has obtained one band of DNA

fragments. By comparing with the Thermo Scientific GeneRuler 1kb DNA ladder in the

lane 1, we approximate the band is 10 000 bp in size. However, the band is slightly

blurred. This might the result of contamination during the procedure of extracting DNA.

The Pipette tip that has been used might contaminated with bacteria or chemical. The

pipette tip should have been directly immersed first in the chemical without touching any

surface. Some of the chemical used such as ethanol is also contaminated during the

preparation. Apart from that, another possible error might arise from the well of the gel

electrophoresis. The comb of the electrophoresis might be inserted wrongly or the gel is

disturbed during the removal of the comb. As a result, the DNA sample unable to come

out very well.

3. Briefly describe what DNA ladders are and how they were generated.

DNA ladders are common reagent composed of known DNA sizes that used to

determine the size of the sample DNA fragment. The sample DNA fragment will be

separated by electrophoresis along with the DNA ladder according to the band of known

sizes.

For example, if the insertion has ten tandem repeats of a 100 bp, we will obtain 100

bp DNA ladder with fragments from 100 bp to 1000 bp. Cloning, PCR and Partial

restriction with restriction enzyme are used to produce DNA ladder. DNA fragments of

100 bp with unique restriction site at both ends are self-ligated to create a tandem
repeat. Once being cloned, the tandem repeat was rapidly amplified by PCR. Tandem

repeat of a DNA fragment is inserted into a plasmid. In each junction of repeat units,

there are the same unique restriction site. A partial restriction digestion of this plasmid

produces a ladder containing multimers of the repeated DNA fragment (Lan, V. T. T.,

Loan, P. T. T., Duong, P. A. T., Thanh, L. T., Ha, N. T., & Thuan, T. B., 2012).

4. In general, ethidium bromide (EtBr) is used to detect DNA during gel

electrophoresis. Explain in detail the mechanism of detection using ethidium

bromide.

To visualise the DNA fragments in agarose gel, the ethidium bromide is usually used

as dye. Under UV light, the excitation and fluorescence emitted by EtBr at maximum

500- 590 nm. This protocol should be performed rapidly to prevent the DNA to degrade

by the long exposure of the UV light. Apart from it, EtBr also a hazardous waste and

should be discarded safely.

After the exposing in the UV light, electrons in the aromatic ring of the ethidium

molecule are activated. The light energy is released as the electrons return to the

ground state. EtBr is an intercalating agent which resembles a DNA base pair. EtBr

works by intercalating itself in the DNA molecule depending with concentration of the

EtBr. Thus, the fluoresces emitted by EtBr within DNA molecule. In addition, EtBr

enable to absorb energy from nucleotides excited by absorbance of 260 nm radiation. It

re-emits this energy as yellow or orange light with 560 nm absorbance. We are allowed

to observe the DNA fragments based on the intensity. Because of the positive charged,

EtBr able to reduce the DNA migration rate by 15%.

Conclusion

In this practical laboratory, we learnt to practice an appropriate protocol to extract

DNA from the soldier termites of Coptotermes gestroi. To get the DNA, the head

area of the Coptotermes gestroi is cut as it is easier to breakdown the cell because

the exoskeleton is softer compared to other regions. If other than head region is

used, we might not see the band in the gel electrophoresis. The result of the gel

electrophoresis is less satisfied as our sample is contaminated during the DNA

extraction. We should redo the procedure and practice precaution to get the best

result.
References
Avise, J. C. (1974). Systematic value of electrophoretic data. Systematic Biology, 23(4), 465-481.

Lan, V. T. T., Loan, P. T. T., Duong, P. A. T., Thanh, L. T., Ha, N. T., & Thuan, T. B. (2012). Straightforward
procedure for laboratory production of DNA ladder. Journal of nucleic acids.

Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H. (2012). Agarose gel electrophoresis for the separation
of DNA fragments. JoVE (Journal of Visualized Experiments), (62), e3923.

Scheffrahn, R. H., & Su, N. Y. (2000). Asian subterranean termite, Coptotermes gestroi (= havilandi)
(Wasmann)(Insecta: Isoptera: Rhinotermitidae). Extension document EENY-128. Institute of Food
and Agricultural Sciences, University of Florida, Gainesville, FL.

Yeap, B. K., Othman, A. S., & Lee, C. Y. (2011). Genetic analysis of population structure of Coptotermes
gestroi (Isoptera: Rhinotermitidae) in native and introduced populations. Environmental
entomology, 40(2), 470-476.