Isolation of DNA Genomes from the Head and Middle Gland of

Silkworms (Bombyx mori L)

Masitta Tanjung

1,2

and Saleha Hannum

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Center of Excellence for Natural Resources-Based Technology, Mangrove and Bio-Resources Group, Universitas

Sumatera Utara, Medan, Indonesia

Keywords: DNA, Silk Glands, Silkworm (Bombyx Mori).

Abstract: The quality and quantity of DNA are very much needed for the molecular analysis of organisms. The

superior quality of DNA samples can be obtained from an appropriate DNA extraction protocol.This

research was conducted to isolate the DNA of silkworm larvae from the head and middle glands. The

silkworms used are from three National silkworm breeding centers, namely Perhutani-Bogor, Bili-Bili and

Soppeng. Organ samples taken are the head and middle silk glands. Samples are crushed and put into

extraction buffers using a Mini Kit (Promega, Madison, WI, USA) in accordance with established

procedures.The measurement of DNA quantity was carried out by the spectrophotometric method using a

spectrophotometer at wavelengths (λ) 260 and 280 nm.DNA purity was determined by calculating the

absorbance ratio at A260 to A280. DNA isolation showed a very thick band (high concentration) and also

firm, this indicated that the results of DNA isolation are very good.DNA samples from the middle silk gland

from the Bogor region were not obtained because the sample used when extracting was too much hence the

extraction buffer used was insufficient to lyse lipids or compounds in the silkworm glands. DNA purity

ranges from 1.75-2.2 with an average purity of 1.95 and the concentration of isolated DNA reaches 37.000

ng / µl.

1 INTRODUCTION

Changes in the nucleotides that make up DNA can

cause genetic diversity. This change can affect the

phenotype of an organism and can affect an

individual's reaction to its environment. This genetic

diversity can occur because of the recombination,

mutation and migration of genes from one place to

another. Nucleotides play a central role in many

cellular processes, including regulation of

metabolism and storage and utilization of genetic

information (Bowater & Gates, 2015). According to

Rao & Hodgkin (2002), genetic diversity can occur

at three levels: (1) ecosystem diversity (species

community and environment), species diversity

(species richness) and genetic diversity (variations in

genes and genotypes).

Genetic diversity begins with the extraction and

purification of DNA. DNA extraction and

purification is the process of separating DNA from

other cell components. Extraction to obtain high-

quality DNA as a condition that must be met in

molecular analysis. DNA extraction and purification

are also one of the success factors in DNA

amplification that will be used in genetic character

analysis. Hybridization programs require genetic

distance, genetic character and not geographical

diversity (Maqbool et al., 2015). A good extraction

must be supported by the results of the quantity of

DNA extract. The measurement of DNA quantity is

using a spectrophotometer at wavelengths (λ) 260

and 280 nm. DNA purity was determined by

calculating the absorbance ratio at A260 to A280

(A260: A280 ratio). DNA molecules are said to be

pure if the absorbance ratio ranges from 1.8 to 2.0.

Information on DNA isolation of the Silkworm

genome identified by microsatellite markers can be

used for breeding programs in encouraging bivoltine

silk production (Bukhari et al., 2019).

DNA isolation can also be used to study the

properties of DNA in certain parts of the body. In

silkworm cocoons (Bombyx mori), fat DNA has

been studied. Pupa body fat DNA is separated into

three components called α-DNA, β1-DNA, and β2-

418

Tanjung, M. and Hannum, S.

Isolation of DNA Genomes from the Head and Middle Gland of Silkworms (Bombyx mori L).

DOI: 10.5220/0010200000002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 418-421

ISBN: 978-989-758-556-2

DNA by the Kieselguhr Albumin Column

Chromatography (MAK) method. All of these DNA

classes are demonstrated as pure DNA, not

contaminated or hybridized with RNA, because it is

positive for the diphenylamine reaction, sensitive to

DNase, resistant to RNase, and includes thymidine-

6-3H but not uridine-5- 3j. The GC content was

calculated from a Tm value of around 38% for these

three components, almost the same as the total

DNA. But the α-DNA molecular weight, which is

roughly calculated from the sedimentation

coefficient on centrifugation gradient sucrose

density, is several times greater than β1-DNA.In the

cocoon stage, body fat DNA consists mainly of β1-

and β2-DNA with small amounts of α-DNA,

whereas in the larval stage, only consists of α-DNA.

Typical body fat DNA types were observed in silk

gland larvae, and in adult pupa tissues such as

integuments, muscles, and gonads. On the other

hand, cocooned fat DNA is detected in degenerating

tissue or in degeneration processes, such as silk

glands, cocoons and midgut. These facts show that

β1- and β2-DNA are degradation products of α-

DNA (Chinzei, 1974).

Silk glands in the larval stage of silkworms,

producing thread as silk material to form a cocoon.

Silk glands are divided into three parts: anterior,

middle and posterior silk glands, each of which

plays a different role in silk secretion. Two new

proteins are identified in the middle silk gland, and

to a lesser extent in the posterior gland, which is

thought to be involved in the regulation of

proteolytic activity and protection of silk proteins

from degradation (Hou et al., 2007).

2 MATERIALS AND METHODS

The silkworms used originated from three National

silkworm breeding centers, which is Pusat

Penelitian dan Pengembangan Hutan Bogor, Balai

Perhutanan Sosial dan Kemitraan Lingkungan

(BPSKL) of the Sulawesi, Bili-bili and Soppeng

region, Sulawesi Selatan. The research begins by

hatching and rearing silkworms until the caterpillar

reaches instar V.

2.1 DNA Isolation

Silkworm head and glands are used as samples for

DNA isolation of the genome. Samples are crushed

and put into extraction buffers using a Mini Kit

(Promega, Madison, WI, USA) in accordance with

established procedures. Samples of silkworms that

have been homogenized are incubated for 15

minutes at 65

C then for 5 minutes at 29-30

Subsequently added 200 μl of protein precipitation

solution and applied vortex for 5 seconds then

centrifuged at 13000 x g for 3 minutes.The

supernatant was taken and put into a micro-tube

containing 600 μl of isopropanol and then

centrifuged at a speed of 13,000 x g for 2 minutes.

The supernatant was removed and the pellet was

washed using 600 µl - 70% ethanol and centrifuged

at a speed of 13,000 x g for 2 minutes. The

supernatant is removed and then dried, then add 50

µl of DNA rehydration solution, applied vortex for 5

seconds then 0.5 µl of RNAse solution is added.

DNA was incubated for 15 minutes at 37

C. DNA

can be incubated at 4

C for 24 hours for further

work. The results of DNA extraction are stored at -

C until it used. DNA concentrations were

calculated using a nano-photometer (IMPLEN,

Munich, Germany, serial no. 6042). Next, DNA was

analyzed by electrophoresis (SCIE-PLAS. Ltd,

Cambridge, England) on 1.2% agarose gel stained

with 1 µl ethidium bromide. The electrophoresis

results were visualized using Gel Doc (Uvitec,

Cambridge, Serial no. 13200263) under UV light

with a wavelength of 303 nm.

3 RESULTS AND DISCUSSION

3.1 Genomic Isolation and

Measurement of DNA

Concentration

The results of silkworm DNA isolation originating

from 3 units of National Natural Silkworm-breeding,

namely Perhutani Bogor, Bili-bili and Soppeng, and

the isolated parts are the head and middle silk glands

can be seen in Figure 1.

Genome isolation showed a very thick band

(high concentration) and also firm, this showed that

isolation of the genome using the Promega kit can

already isolate DNA well. According to Bukhari et

al, (2019), for molecular analysis of any organism,

superior quality DNA samples obtained from a

suitable DNA extraction protocol is needed. In the

Bg2 sample, there was no genomic or DNA band

because the sample used when extracting was too

much hence the extraction buffer used was

insufficient to lyse lipids or compounds in the

silkworm glands resulting in failure to extract the

DNA (the lysis reaction failed).DNA extraction

and purification is basically a series of processes

of separating DNA from other cell components.

Isolation of DNA Genomes from the Head and Middle Gland of Silkworms (Bombyx mori L)

419

Figure 1: The results of the extraction of silkworm DNA

genomes from 3 National silkworm nurseries. Bg1:

Silkworm comes from the Bogor area, isolating the sample

from the head; Bg2: Silkworm comes from the Bogor

region, isolating the sample from the gland; Bi1:

Silkworm comes from Bili-bili, isolating the sample from

the head; Bi2: Silkworm comes from Bili-bili, isolating

samples from glands; SP1: Silkworm comes from

Soppeng, isolating the sample from the head; SP2:

Silkworm comes from Soppeng, isolating samples from

the glands.

Extraction to obtain high quality DNA is a basic

principle that must be fulfilled in molecular analysis

and is one of the success factors in DNA

amplification that will be used in genetic character

analysis. According to (Kawamotoa et al., 2019),

incorrect genome assembly sometimes leads to

incorrect gene prediction. Good extraction is

supported by the results of the number of DNA

extracts obtained through spectrophotometric

methods using spectrophotometers at wavelengths

(λ) of 260 and 280 nm. UV analysis on DNA has

become a benchmarking method for rapid

quantification and testing of sample purity for

various purposes (Pachchigar et al., 2016). The

purity and concentration of DNA that has been

isolated can be seen in Table 1.

Table 1: Quantitative analysis of Bombyx mori L.

silkworm DNA genomes from 3 National silkworm

nursery sites.

No Sample Code

Absorbance

(A260/280)

Concentration

(ng/ul)

1 Bg1 2,2 13.000

2 Bi1 1,9 30.000

3 Bi2 2,0 37.000

4 Sp1 1,75 2.360

5 Sp2 1,9 7.120

Based on Table 1, it can be seen that DNA Purity

ranges from 1.75-2.2 with an average purity of 1.95

with the conclusion that the isolated DNA is pure

(Wilson & Walker, 2010). DNA purity was

determined by calculating the absorbance ratio at

A260 to A280 (A260: A280 ratio). DNA molecules

are said to be pure if the absorbance ratio ranges

from 1.8 to 2.0 (Sambrook et al., 1989). DNA

concentrations were very high, especially in the Bi1

sample code, which was 37000 ng/ul, while the

lowest in Sp2 was 7.120 ng/ul, but this concentration

was very sufficient to continue further analysis.DNA

concentrations that are too low will produce

fragments that are very thin on the gel or even not

visually visible, on the contrary, DNA

concentrations that are too high will cause the

fragments to appear thick hence it is difficult to

distinguish between fragments from other fragments.

The concentration of DNA extraction results is

influenced by 2 factors: the extraction rate at the

time of extraction and the composition of the buffer

lysis addition. The extraction speed factor is the

most influential factor because it is present in the

stages of cell lysis and precipitation.The application

of isolation is for structural analysis and functional

analysis of the desired gene. The application of

isolation by combining labels can be used to monitor

enzymatic reactions, nucleic acid processing and

hybridization experiments. The interaction of

proteins and nucleic acids plays a major role in all

aspects of gene expression. Proteins in the larvae

and silk glands, DNA and RNA in the larvae and

silk glands are separated and presented with the help

of PAGE, SDS-PAGE, and PFGE respectively. This

has been a pioneer in the field of silkworm

sericulture (Brindha et al., 2012).

The silk glands of Bombyxmori are very

different tissues where DNA replication continues

without cell division during larval development.

DNA polymerase-delta activity is increased in the

posterior and middle silk glands during the

development period, reaching maximum levels in

the middle silk gland of the fifth instar larvae.There

are many ways to preserve insect specimens that will

protect their DNA from degradation during the

collection period for use in molecular genetic

studies. However, techniques vary among groups of

insects, suggesting that preservation must be carried

out before working at the molecular level with large

numbers of individuals from certain protein groups.

Protein in the silk glands and at each larval stage is

estimated quantitatively.

Bg2

Bi1

Bi2

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

420

4 CONCLUSION

The quality and quantity of DNA is very much

needed for the molecular analysis of organisms. The

superior quality of DNA samples can be obtained

from an appropriate DNA extraction protocol.

Isolation of silkworm DNA (B. mori L.) can be done

from the head and middle silk glands.In silk-gland

samples must be adjusted to the concentration of the

buffer because if there is not enough amount of

extraction buffer used, it will fail the process of lipid

lysis or compounds in the silkworm gland. DNA

purity ranged from 1.75-2.2 with an average purity

of 1.95 and the concentration of isolated DNA

reached 37,000 ng / µl.

ACKNOWLEDGEMENT

The author would like to thank the University of

Sumatera Utara for funding this research with a

contract through the University of Sumatera Utara

Research Institute Adjusted to the TALENTA

Research University of Sumatera Utara Contract

Year 2019 Fiscal Year Number:

4167/UN5.1.R/PPM/2019, April 1, 2019

Acknowledgments were also conveyed to the

Faculty of Mathematics and Natural Sciences for

facilitating the laboratory and thanking all the parties

involved.

REFERENCES

Bowater, R. P., & Gates, A. J. (2015). Nucleotides:

Structure and Properties. John Wiley & Sons.

https://doi.org/10.1002/9780470015902.a0001333.pub

Brindha, S. ., Maragathavalli, S., CGangwar, S. ., & c

Annadurai, B. (2012). Isolation, Purification And

Kinetics Of Deoxy Ribonucleic Acid At Different

Stages Of Feeding In Bombyx Mori G.J.B.B. Global

Journal Of Bio-Science & Biotechnology, 1(1), 22–28.

Bukhari, R. M., Sharma, R. ., Bali, R. K., Salgotra, R. H.,

& Shah. (2019). A modified efficient protocol for

DNA extraction in Silkworm (Bombyx mori L.).

Journal of Entomology and Zoology Studies, 7(1),

867–870.

Chinzei, Y. (1974). Biochemical properties of fat body

DNA of the silkworm, Bombyx mori. Journal of

Insect Physiology, 20(12), 33–46.

Hou, Y., Xia, Q., Zhao, P., Zou, Y., Liu, H., Guan, J.,

Gong, J., & Xiang, Z. (2007). Studies on middle and

posterior silk glands of silkworm (Bombyx mori)

using two-dimensional electrophoresis and mass

spectrometry. Insect Biochem Mol Biol, 37(5), 486–

496.

Kawamotoa, M. ., Jourakub, A., Toyodac, K., Yokoib, Y.,

Minakuchic, S., Katsumaa, A., Fujiyamac, T.,

Kiuchia, K., Yamamotob, T., & Shimadaa. (2019).

High-quality genome assembly of the silkworm,

Bombyx mori. Insect Biochemistry and Molecular

Biology, 107, 53–62.

Maqbool, A. H. U., Dar, M., Ahmad, G. N., Malik, G., &

Zaffar, S. A. M. (2015). Genetic divergence in some

bivoltine silkworm (Bombyx mori L.) breeds.

Scientific Research and Essays, 10(11), 381–385.

Pachchigar, K. P., Khunt, A., & Hetal, B. (2016). DNA

quantification Methods and Utility. Agricultural

University, Sardarkrushinagar, 17(4), 379–386.

Rao, V. ., & Hodgkin, T. (2002). Genetic diversity and

conservation and utilization of plant genetic resources.

Plant Cell, Tissue and Organ Culture, 68, 1–19.

Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989).

Molecular cloning: a laboratory manual. University

of Texas South Western Medical Center.

Wilson, K., & Walker, J. (2010). Principles and

Techniques of Biochemistry and Molecular Biology

(7th ed.). Cambridge University Press.

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