Searching for the best agarose candidate from genus Gracilaria，Eucheuma，Gelidium and local brands

Ferry Efendi，Retno Handajani，Nursalam NursalamFaculty of Nursing，Airlangga University，Surabaya，Indonesia

2Department of Biochemistry Medicine，Faculty of Medicine，Airlangga University，Surabaya，Indonesia

3Institute of Tropical Disease，Airlangga University，Surabaya，Indonesia

Searching for the best agarose candidate from genus Gracilaria，Eucheuma，Gelidium and local brands

Ferry Efendi1*，Retno Handajani2，3，Nursalam Nursalam1
1Faculty of Nursing，Airlangga University，Surabaya，Indonesia

2Department of Biochemistry Medicine，Faculty of Medicine，Airlangga University，Surabaya，Indonesia

3Institute of Tropical Disease，Airlangga University，Surabaya，Indonesia

ARTICLE INFO

Article history：

Accepted 25 Jun 2015

Available online 7 Aug 2015

Agarose

Eucheuma

Gracilaria

Gelidium

Local brands

Indonesia agar

Objective：To explore the potential of local agar of genus Gracilaria，Eucheuma，Gelidium and local brands as an alternative for imported agarose for DNA electrophoresis，and to examine their ability related to separation and migration of DNA fragments in DNA electrophoresis.

Methods：Their performance at various concentrations were compared via an experimental study with a specific brand of imported commercial agarose used in molecular biology research.The measured variables were separation and migration during electrophoresis of a DNA fragment.

Results：The local agar genus Gracilaria gigas，Gelidium，brand“B”and brand“S”could separate DNA fragments at a concentration between 1%and 2%，with an optimum concentration of 2%w/v，as good as a specific brand of imported commercial agarose.

Conclusions：Their performance were very close to that of commercial agarose and can still be improved by further agar purification as well as by pH and sulfur control.

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2015.06.009

1.Introduction

Major advances in molecular biology characterization cannot be separated from the availability of polysaccharides from seaweed or algae［1］.Most DNA separation and analysis require agarose gel electrophoresis［2，3］.Problems arising in this method include poor accessibility and high costs，mainly in the case of less developed and developing countries，considering the fact that most of the materials are imported.This problem needs to be considered in the majority of developing countries with regard to confirming diagnoses.One of the imported materialsis agarose，a polysaccharide polymer extracted from seaweed that is frequently used in biochemistry for gel electrophoresis［3］.A considerable amount of literature has been published on agarose gel electrophoresis.These studies have shown that agarose gel electrophoresis is the most effective way to separate DNA fragments［3-5］.

Indonesia，which is considered the seaweed producer of the world，produces tons of various types of seaweed［6］.The centers of seaweed production spread from the east to the west of Indonesia in order to supply the world demand.For instance，datain2010statedthatIndonesianseaweed productionongenusGracilariaandEucheumacottonii（E.cottonii）was around 3 082 112 tons［7］.These genus have the potential to be used in DNA separation，but have not yet been explored.

The objective of this research was to examine the separation and migration capacity of DNA fragment by using the local agar of genus Gracilaria，Eucheuma，Gelidium and local brands.Later，those properties are compared to a specific brand of imported commercial agarose.This research was aimed at anexploration of a new agarose candidate for gel electrophoresis by using the local agar from Indonesian seaweed.

2.Materials and methods

The agarose materials used in this research were:（1）agarose electrophoresis grade，obtained from MP Biomedicals，California，United States of America with catalog No.820721.It was used as a control.This white agarose powder was ordered from an international supplier，and further information about this product was available at www.mpbio.com；（2）local produced agarose materials，including:a.agar powder，consisting of genus Gracilaria gigas（G.gigas），Gelidium，Eucheuma spinosum and E.cottonii.The powder used was still under research and development and had not been commercialized as of the time of this writing；b.rods agar，consisting of genus Gelidium；c. commercial agar of brands“S，B，C and N”；d.agar sheets，consisting of Nico nori，Nori and Seaweed nori.

Both a and b agars were obtained from a local seaweed factory，located in Surabaya，Indonesia，and the genus was verified by the quality control division of the factory，while c and d agars were obtained from a local market in Surabaya，Indonesia.

Agar powder was purified by heating and centrifugation.The solvents used consisting of a Tris Borate EDTA buffer（TBE 0.5×）prepared by diluting 5×TBE.Each different concentration of agar（0.5%，1.0%，1.5%and 2.0%）was heated and then transferred into a centrifuge tube for 5 min at 3000 r/min.By this way，particles with high molecular weights were expected in the bottom of centrifuge tube.After cooling，the agar was readily formed and was used for the next step.A third-quarter part of the top freeze agar was collected and heated in a microwave for 1 min.About 20 mL of it was later mixed with ethidium bromide of 2μL and molded into a gel tray.It was ready to use for electrophoresis after gel freezes.This research was conducted at the molecular biology laboratory，Institute of Tropical Disease，Airlangga University，Surabaya，Indonesia.

3.Results

Table 1 Gel strength of the tested agars in experiment.

Table 1 shows the summary of the performance of the different tested agars.Here，we conducted a visual assessment and expert judgment was provided by a second author and was confirmed by another author and a laboratory assistant.As depicted in Table 1，only agar powder of genus G.gigas，Gelidium and market agar from brand“S”and brand“B”showed gel strength.In addition，the gel was colorless and could be observed easily whereas，others exhibited less gel strength and the gel was not clear and transparentenoughincolor，whicheventuallycaused difficulty in regard to observation.The highest quality selected agar was used for electrophoresis and was compared with a specific brand of imported commercial agarose as a control.

3.1.G.gigas

Electrophoresis result was generated from agar powder of genus G.gigas by using DNA 100 bp and ØX 174-Hae III Digest markers（Figure 1）.

3.2.Gelidium

Electrophoresis result came from agar powder genus of Gelidium using DNA 100 bp and ØX 174-Hae III Digest markers（Figure 2）.

3.3.Agar brand“B”

Electrophoresis result was generated from agar brand“B”by using DNA 100 bp and ØX 174-Hae III Digest markers（Figure 3）.

3.4.Agar brand“S”

Electrophoresis result was generated from agar brand“S”by using DNA 100 bp and ØX 174-Hae III Digest markers（Figure 4）.

Fromtheaboveselectedagars，weconducteda proximate analysis.Proximate analysis included analysis of moisture，ash，crude protein，crude lipid，crude fiber，carbohydrates，nitrogen free extract and metabolic energy，as shown in Table 2.

Comparation results in electrophoresis of the imported agarose and local agar based on migration and resolution are shown in Figures 5-8.

Table 2 Results of proximate analysis（%）.

4.Discussion

As shown in Figure 5，both imported and local agar genus Gracilaria could separate very well and migrate DNA.However，the resolution of the imported agarose was brighter and sharper when compared with G.gigas in regard to both markers（100 bp and ØX 174-Hae III Digest）.This condition can also be seen in Figures 6-8.Local agarose separated and migrated the DNA fragment，but the resolution was not very sharp.In general，the results showed that local agar could be a good agarose alternative despite the fact that its separation ability and separation resolution were not very sharp.Within the tested concentration range，local agar could separate the DNA fragments at all concentrations，and the best separation and resolution were obtained at concentrations of 1%-2%.Some of the shortcomings of the market agars mainly lay in the separation of DNA fragments when they were used at a low concentration（0.5%）.In general，at a concentration of 2%，the separation of DNA fragments was found to be better than at another three lower concentrations.There were some factors affecting the separation and migration ability of the DNA fragments，which included agarose concentrations，the size of DNA fragment，the voltage applied，the type of agarose，the presence of ethidium bromide and the electrophoresis buffer［4］.The effects of concentrations on the electrophoresis results have been documented in other studies，where it has been commonly shown that the concentration is dependent on the DNA molecular weight，which varies from 0.5%to 1.5%［8］.However，general concentrations have been reported at 1%for many applications［9］.In this study，we found that at concentrations between 1%and 2%，the local agar exhibited the capability to run and migrate the DNA fragments with 100 bp and ØX 174-Hae III Digest markers.

The cause of differences in the electrophoresis results may have been the presence of undesirable components in the local agarose that affected the separation of fragments，such as preservatives and dyes.In addition，the sulfur content and pH of each local agarose were not controlled in this study，which may have significantly affected the results.It should be noted that the electrophoresis results were affected by pH and denaturants that were present in the medium［10］.The proximate analysis showed that the selected agar contained crude proteins ranging from 1.3%to 2.1%（Table 2）.

This study describes the quality of DNA fragment separation by using a local agar and shows the potential of some local agars to substitute for imported agarose.These local agars offer substantial advantages，especially in terms of low cost and easy accessibility.The quality of separation and the separation resolution from these agars can still be improved by controlling sulfur content，pH and of course the presence of certain undesirable materials.These factors will be explored further in the future.The local agar of genus G.gigas as well as Gelidium，brand“B”and brand“S”could separate DNA fragments and migrate well by using 100 bp and ØX 174-Hae III Digest markers.Better visible resolution was observed in the aforementioned agars at a concentration between 1%and 2%and at an optimum concentration of 2%w/v.Further research on local agar characterization，standard purification techniques and other factors affecting agarose resolution is still necessary.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

We would like to thank Dr.Muhammad Roil Bilad，as a researcher at Nanyang Technological University，Singapore，for his critical comments and editing assistance in writing the manuscript.This work was supported by the Directorate General of Higher Education（Dirjen Dikti），Ministry of National Education of Indonesia through the“Penelitian Hibah Bersaing”scheme（Grant No.436/J）3.2/PG/2008）.

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21 May 2015

Ferry Efendi，Faculty of Nursing，Airlangga University，Surabaya，Indonesia.

Tel:+62 8998417244

E-mail:fefendi@indonesiannursing.com

Peer review under responsibility of Hainan Medical University.

Foundation Project:Supported by the Directorate General of Higher Education（Dirjen Dikti），Ministry of National Education of Indonesia through the“Penelitian Hibah Bersaing”scheme（Grant No.436/J）3.2/PG/2008）.

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