ORIGINAL_ARTICLE
Targeting Colorectal Cancer Cell Lines Using Nanobodies; AgSK1as a Potential Target
Background: Colorectal cancer is the third most common type of aggressive cancers. Chemotherapy, surgery,and radiotherapy are the common therapeutic options for treating this cancer. Due to the adverse side-eff ects of these methods, immunotherapy is considered as an appropriate alternative therapeutic option. Treatment through the application of monoclonal antibodies is considered as a novel alternative therapeutic method for cancers. The variable fragments of the antibodies’ heavy chain or VHHs have a wide application in molecular biology and biotechnology. VHHs are compatible with the phage display technology which allows rapid and high throughput screening for antibodies isolation.Objectives: We aimed to use naive VHH phage library to isolate a specifi c nanobody against colorectal tumorassociated antigen; the AgSK1.Materials and Methods: In this research, naive VHH phage library was panned against two colorectal cell lines;Ls174T and HT29 expressing diff erent levels of AgSK1 tumor associated marker. The high affi nity binders were selected and subcloned for higher expression levels of the VHH. The affi nity and specifi city of the isolated VHH were tested using ELISA. The reactivity of the VHH toward cancer cells was analyzed by competitive ELISA applying sera isolated from colorectal cancer patients.Results: Results show that the isolated VHH recognizes and binds to the colorectal cancer cells with a highaffi nity. Moreover, the isolated nanobody is able to compete with the antibodies in the patient sera for the binding to the cancer cells.Conclusions: Results suggest that this nanobody has a specifi c reaction toward colorectal cells and can be used for further investigation on the tumor associated antigens or production of mimotopes useful for immunotherapy.
https://www.ijbiotech.com/article_47020_05e528e8877b1e8f8d84423fbbf03898.pdf
2017-04-01
78
86
DOI:10.15171/ijb.1472
AgSk1
Cell-panning
colorectal cancer
VHH nanobody
Seyed Khalil
Rashidi
m.kh.rashidi@gmail.com
1
Department of Biology, Faculty of Basic Science, Shahed University, Tehran, Iran
AUTHOR
Seyed Latif
Mousavi Gargari
slmousavi@shahed.ac.ir
2
Department of Biology, Faculty of Basic Science, Shahed University, Tehran, Iran
LEAD_AUTHOR
Walead
Ebrahimizadeh
w_ebrahimizadeh@hotmail.com
3
Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran.
AUTHOR
ORIGINAL_ARTICLE
TiO2 Nanoparticles as Potential Promoting Agents of Fibrillation of α-Synuclein, a Parkinson’s Disease-Related Protein
Background: In recent years, nanomaterials have been widely used in large quantities which make people bemore frequently exposed to the chemically synthesized nanoparticles (NPs). When NPs are introduced intoan organism, they may interact with a variety of cellular components with yet largely unknown pathologicalconsequences.Objective: It was found that NPs enhance the rate of protein fi brillation in the brain by decreasing the lag time for nucleation. Protein fi brillation is implicated in the pathogenesis of the several neurodegenerative diseases such as Parkinson’s disease (PD). α-Synuclein (αS) is natively an unfolded protein which is involved in the pathogenesis of PD. In the present study, we have analyzed the eff ects of three diff erent NPs on αS fi brillation.Materials and Methods: αS protein expression and purifi cation was done and fi brils formation was inducedin the absence or presence of the three types of NPs (i. e., TiO2, SiO2, and SnO2). The enhancement of thefl uorescence emission of Thiofl avin T (ThT) and transmission electron microscopy (TEM) were used to monitor the appearance and growth of the fi brils. The adsorption of αS monomers on the surface of NPs was investigated by tyrosine fl uorescence emission measurements.Results: We found that TiO2-NPs enhances αS fi bril formation even at a concentration of 5 μg.mL-1, whilethe two other NPs show no signifi cant eff ect on the kinetics of the fi brillation. Intrinsic tyrosine emissionmeasurement has confi rmed that the TiO2-NPs interact with αS fi brillation products. It is suggested that TiO2-NPs may enhance the nucleation of αS protein that leads to protein fi bril formation.Conclusion: The fi brillization process of αS protein is profoundly aff ected by the presence of TiO2-NPs. Thisfi nding unveils the neurotoxicity potential of the TiO2-NPs, which may be considered as a probable risk for PD.
https://www.ijbiotech.com/article_47057_fdd72f4a3a02a31eeba0ea96ff06bfaa.pdf
2017-04-01
87
94
DOI:10.15171/ijb.1519
α-Synuclein (αS)
Nanoparticles (NPs)
Parkinson’s disease (PD)
Titanium Dioxide Nanoparticles (TiO2-NPs)
Soheila
Mohammadi
soheila.mohammadi@modares.ac.ir
1
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115-175 Iran
AUTHOR
Maryam
Nikkhah
m_nikkhah@modares.ac.ir
2
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115-175 Iran
LEAD_AUTHOR
ORIGINAL_ARTICLE
Biosynthesis of Silver Nanoparticles Using Pine Pollen and Evaluation of the Antifungal Efficiency
Background: Nanoparticles have been applied to medicine, hygiene, pharmacy and dentistry, and will bring significant advances in the prevention, diagnosis, drug delivery and treatment of disease. Green synthesis of metal nanoparticles has a very important role in nanobiotechnology, allowing production of non-toxic and eco-friendly particles.Objectives: Green synthesis of silver nanoparticles (AgNPs) was studied using pine pollen as a novel, cost-eff ective, simple and non-hazardous bioresource. The antifungal activity of the synthesized AgNPs was investigated in vitro.Materials and Methods: Biosynthesis of AgNPs was conducted using pollen of pine (as a novel bioresource) acting as both reducing and capping agents. AgNPs were characterized using UV-visible spectroscopy, X-ray diff raction and transmission electron microscopy. In evaluation for antifungal properties, the synthesized AgNPs represented signifi cant in vitro inhibitory effects on Neofusicoccum parvum cultures.Results: Pine pollen can mediate biosynthesis of colloidal AgNPs with an average size of 12 nm. AgNPs were formed at 22C and observed to be highly stable up to three months without precipitation or decreased antifungal property. AgNPs showed signifi cant inhibitory eff ects against Neofusicoccum parvum.Conclusion: The first report for a low-cost, simple, well feasible and eco-friendly procedure for biosynthesis of AgNPs was presented. The synthesized AgNPs by pine pollen were nontoxic and eco-friendly, and can be employed for large-scale production. The nanoparticles showed strong eff ect on quantitative inhibition and disruption of antifungal growth.
https://www.ijbiotech.com/article_47018_302a7cc4257236efc3f7f99ffd511d00.pdf
2017-04-01
95
101
DOI:10.15171/ijb.1436
Diagnosis
low-cost technology
colloidal silver nano particles
Pollen
Mehrdad
Khatami
mehrdad7khatami@gmail.com
1
School of Medicine, Bam University of Medical Sciences, Bam, Iran.
LEAD_AUTHOR
Seyed Mojtaba
Mortazavi
m.khatami@mubam.ac.ir
2
School of Medicine, Bam University of Medical Sciences, Bam, Iran.
AUTHOR
Zeinab
Kishani Farahani
dr.farahani@mubam.ac.ir
3
Research and Development Center, Taleghani Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran
AUTHOR
Abbas
Amini
abbasust@gmail.com
4
Institute for Infrastructure Engineering, Western Sydney University, Kingswood Campus, Locked Bag 1797, NSW 2751, Australia
AUTHOR
Elham
Amini
elham.amini62@gmail.com
5
Research Center for Tropical and Infectious Diseases, Kerman University of Medical Sciences, Kerman, Iran
AUTHOR
Hossein
Heli
hheli7@yahoo.com
6
Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
AUTHOR
ORIGINAL_ARTICLE
Production of Marker-free Transgenic Rice (Oryza sativa L.) with Improved Nutritive Quality Expressing AmA1
Background: Rice seed proteins are lacking essential amino acids (EAAs). Genetic engineering off ers a fast and sustainable method to solve this problem as it allows the specifi c expression of heterologous EAA-rich proteins. The use of selectable marker gene is essential for generation of transgenic crops, but might also lead to potential environmental and food safety problems. Therefore, the production of marker-free transgenic crops is becoming an extremely attractive alternative and could contribute to the public acceptance of transgenic crops.Objectives: The present study was conducted to examine whether AmA1 can be expressed specifi cally in rice seeds, and generate marker-free transgenic rice with improved nutritive value.Materials and Methods: AmA1 was transferred into rice using Agrobacterium-mediated co-transformation system with a twin T-DNA binary vector and its integration in rice genome was confi rmed by southern blot. Transcription of AmA1 was analyzed by Real-Time PCR and its expression was verifi ed by western analysis. Protein and amino acid content were measured by the Kjeldahl method and the high-speed amino acid analyzer, respectively.Results: Five selectable marker-free homozygous transgenic lines were obtained from the progeny. The expression ofrecombinant AmA1 was confi rmed by the observation of a 35 kDa band in SDS-PAGE and western blot. Compared to the wild-type control, the total protein contents in the seeds of fi ve homozygous lines were increased by 1.06~12.87%. In addition, the content of several EAAs, including lysine, threonine, and valine was increased signifi cantly in the best expressing line.Conclusions: The results indicated that the amino acid composition of rice grain could be improved by seed-specific expression of AmA1.
https://www.ijbiotech.com/article_47059_456f3c5c20d6fbf4fac60b76edb1bd01.pdf
2017-04-01
102
110
DOI:10.15171/ijb.1527
AmA1 gene
Co-transformation
Essential amino acid
Selectable marker-free
Rice
Ming
Xu
xmfau@163.com
1
Crop Quality Institute, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
AUTHOR
Shuai
Zhao
1258637127@qq.com
2
Crop Quality Institute, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
AUTHOR
Wen
Zhang
823401561@qq.com
3
Crop Quality Institute, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
AUTHOR
Hengjie
Yin
1031976996@qq.com
4
Crop Quality Institute, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
AUTHOR
Xuejuan
Peng
448909691@qq.com
5
CCrop Quality Institute, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
AUTHOR
Zuxin
Cheng
chengzuxin@163.com
6
Crop Quality Institute, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
AUTHOR
Zhijian
Yang
yangzj41@163.com
7
Crop Quality Institute, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
AUTHOR
Jingui
Zheng
jgzheng@fafu.edu.cn
8
Crop Quality Institute, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China
LEAD_AUTHOR
ORIGINAL_ARTICLE
Antimicrobial Activity of Chitosan Film Forming Solution Enriched with Essential Oils; an in vitro assay
Background: The resistance of the bacteria and fungi to the innumerous antimicrobial agents is a major challenge in the treatment of the infections demands to the necessity for searching and fi nding new sources of substances with antimicrobial properties. The incorporation of the essential oils (EOs) in chitosan fi lm forming solution may enhance antimicrobial properties. However, its use as the feeding additive in the poultry nutrition needs to clarify the product’s activity against both pathogen and the useful microbes in the gastrointestinal tract.Objectives: In the present study, we carried out an in vitro investigation and evaluated the antimicrobial activity of chitosan film forming solution incorporated with essential oils (CFs+EOs) against microbial strains including Staphylococcus aureus, Escherichia coli, Enterococcus faecium, Lactobacillus rahmnosus, Aspergillus niger and Alternaria alternate.Material and Methods: In three replicates, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of diff erent treatments including: 1- essential oils (EOs), 2- chitosan fi lm solution (CFs), and 3-chitosan film solution enriched with EOs (CFs+EOs) were determined against above mentioned microbes.Results: The results indicated that the chitosan solution enriched with essential oils (CFs+EOs) is capable of inhibiting the bacterial and fungal growth even at the lowest concentrations. The MIC and MBC for all the antimicrobial agents against Escherichia coli and Staphylococcus aureus were very low compared to the concentrations needed to inhibit the growth of useful bacteria, Lactobacillus rahmnosus and Enterococcus faecium. The antifungal activity of chitosan was enhanced as the concentration of EOs increased in the fi lm solution.Conclusion: Chitosan-EOs complexes are the promising candidate for novel contact antimicrobial agents that can be used in animal feeds.
https://www.ijbiotech.com/article_47015_a02827d1ad49f306b2779d4708e7b63b.pdf
2017-04-01
111
119
DOI:10.15171/ijb.1360
Antimicrobial properties
Bacteria
Chitosan, Chitosan fi lm forming solution
Essential oils
Fungi
kana
Raphael
kanajean@yahoo.fr
1
Department of Animal Productions, Faculty of Agronomy and Agricultural Sciences, University of Dschang, 70 Dschang, Cameroon
AUTHOR
Amir
Meimandi
meimandi@nigeb.ac.ir
2
Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, 14965/161, Iran
LEAD_AUTHOR
ORIGINAL_ARTICLE
Production of Xanthanases by Paenibacillus spp.: Complete Xanthan Degradation and Possible Applications
Background: A number of microorganisms and their enzymes have been reported as xanthan depolymerizers. Paenibacillus species are well-known polysaccharide hydrolyzing bacteria. However, Paenibacillus alginolyticus and Paenibacillus sp.XD are the only species in the genus which are now known to degrade xanthan.Objectives: Complete biodegradation of the xanthan exopolysaccharide is a rarely found capability among microorganisms. The aim of this study is to survey xanthanase producing bacteria with an appropriate bioactivity for the biopolymer degradation under diff erent environmental conditions.Materials and Methods: The bacteria were isolated based on viscosity reduction of the xanthan solution. Bacterial isolates were identifi ed using rep-PCR (repetitive element-based genomic fi ngerprinting) and 16S rDNA sequencing. Xanthanases were characterized by measuring their activity at diff erent temperatures, pH values, and NaCl concentrations. Degradation of other polysaccharides and xanthan degradation products were investigated based on the screening plate method and TLC (thin-layer chromatography), respectively.Results: Six isolates from diff erent Paenibacillus species with a complete xanthan degrading capability were isolated from Urmia Lake. Phylogenetic analysis placed these strains within the genus Paenibacillus with the closest relatives that were found to be P. nanensis, P. phyllosphaerae, P. agaridevorans, P. agarexedens, and P. taohuashanense. These isolates displayed diff erent levels of the xanthan biodegradation activity in temperatures ranging from 15 to 55 °C and pH values from 4 to 11. Xanthanolytic activity was generally prevented in presence of NaCl (> 0.1 mol.L-1). Furthermore, the isolated Paenibacillus spp. could degrade several other polysaccharides including xylan, CMC (carboxymethyl cellulose), starch, alginate, and pectin.Conclusion: Novel strains of the six diff erent Paenibacillus species that were introduced in the present study are able to produce xanthanases with interesting characteristics. In light of the results from this study, special applications, particularly in healthcare, medicine, and the environment is hereby proposed for these enzymes.
https://www.ijbiotech.com/article_47021_72ef80f3663feb36d4490211a24125a7.pdf
2017-04-01
120
127
DOI:10.15171/ijb.1477
Bacterial enzymes
Biodegradation
Paenibacillus spp
Xanthan lyase
Xanthanase
Simin
Ashraf
s.ashraf.micro@gmail.com
1
Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Postal code:1993893973. Iran
AUTHOR
Mohammad Reza
Soudi
soudimr@gmail.com
2
Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Postal code:1993893973. Iran
LEAD_AUTHOR
Parinaz
Ghadam
pghadam@alzahra.ac.ir
3
Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Postal code:1993893973 Iran
AUTHOR
ORIGINAL_ARTICLE
The Increase in Protein and Plasmid Yields of E. coli with Optimized Concentration of Ampicillin as Selection Marker
Background: Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically aff ect the yield of protein and plasmid.Objectives: Alterations of protein expression and plasmid yields of E. coli in diff erent concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized.Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F’. The expression of GFP was verifi ed by SDS-PAGE and fl ow cytometry after induction by IPTG (0.5 mM) and incubation with 0, 100, 200 and 300 μg.mL-1 ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve.Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 fi lter of fl ow cytometry and an extra protein band on SDS-PAGE gel. The fl uorescent intensity of GFP in 0, 100, 200 and 300 μg.mL-1 ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07×109, 3.21×109, 2.32×1010 , 8.11×108, respectively. The plasmid yields were 55 ng.μL-1, 69 ng.μL-1, 164 ng.μL-1 and 41 ng.μL-1, respectively.Conclusion: Protein and plasmid yields of E. coli are variable in diff erent concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration (200 μg.mL-1) was signifi cantly (p < 0.01) higher than other doses.
https://www.ijbiotech.com/article_47019_d2d0e812287ff2bcc9f52389c4411c33.pdf
2017-04-01
128
134
DOI:10.15171/ijb.1467
Ampicillin
Escherichia coli
Plasmid
Protein
Sadegh
Feizollahzadeh
sadeghimmune@yahoo.com
1
Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
AUTHOR
Shirin
Kouhpayeh
kouhpayeh@resident.mui.ac.ir
2
Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
AUTHOR
Ilnaz
Rahimmansh
ilnazrahimmansh@yahoo.com
3
Department of Molecular Biology and Genetics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
AUTHOR
Hossein
Khanahmad
h_khanahmad@med.mui.ac.ir
4
Department of Molecular Biology and Genetics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
AUTHOR
Faezeh
Sabzehei
faezehfafa67@yahoo.com
5
Department of Molecular Biology and Genetics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
AUTHOR
Mazdak
Ganjalikhani-hakemi
mghakemi@med.mui.ac.ir
6
Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
AUTHOR
Alireza
Andalib
andalib@med.mui.ac.ir
7
Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
AUTHOR
Zahra
Hejazi
zahrahejazi88@gmail.com
8
Department of Molecular Biology and Genetics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
AUTHOR
Abbas
Rezaei
rezaei@mui.ac.ir
9
Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 81746-73461 Iran
LEAD_AUTHOR
ORIGINAL_ARTICLE
Biological Removal of the Mixed Pharmaceuticals: Diclofenac, Ibuprofen, and Sulfamethoxazole Using a Bacterial Consortium
Background: The presence of pharmaceuticals at low concentrations (ng to μg) in the environment has become a hot spot for researchers in the past decades due to the unknown environmental impact and the possible damages they might have to the plantae and fauna present in the aquatic systems, as well as to the other living organisms.Objectives: The aim of the present investigation was to develop a bacterial consortium isolated from diff erent origins to evaluate the ability of such a consortium to remove a mixture of pharmaceuticals in the batch system at lab scale, as well as assessment of its resistance to the other micropollutants present in the environment.Material and Methods: Using a closed bottle test, biodegradation of the mixed pharmaceuticals including Diclofenac (DCF), Ibuprofen (IBU), and Sulfamethoxazole (SMX) (at a concentration of 3 mg.L-1 of each drug) by the bacerial consortium was investigated. The test was carried out under metabolic (pharmaceutical was used as the sole source of carbon) and co-metabolic condition (in the presence of glucose). Finally, the ability of the bacterial consortium to resist other micropollutants like antibiotics and heavy metals was investigated.Results: Under the metabolic condition, the mixed bacteria (i.e., consortium) were able to metabolize 23.08% and 9.12% of IBU, and DCF at a concentration of 3 mg.L-1 of each drug, respectively. Whereas, in co-metabolic conditions, IBU was eliminated totally, in addition, 56% of the total concentration of DCF was removed, as well. In both metabolic and cometabolic conditions, removal of SMX was not observed. The selected bacteria were able to resist to most of the applied antibiotics and the used heavy metals, except mercury, where only one strain (S4) was resistant to the later heavy metal. Conclusion: Results suggest that the developed consortium might be an excellent candidate for the application in thebioremediation process for treating ecosystems contaminated with the pharmaceutical.
https://www.ijbiotech.com/article_47061_382f3beaae0a9e6a725a2815ccd0fa9f.pdf
2017-04-01
135
142
DOI:10.15171/ijb.1530
Bacterial consortium
Biodegradation
Co-metabolism
Mixed pharmaceutical
Salima
Aissaoui
aissa.salima@yahoo.fr
1
1Laboratory of Molecular Toxicology, Faculty of Nature and life Sciences, University of Mohammed Seddik Benyahia - Jijel, 98 Ouled Aissa-Jijel 1800-Algeria, Algeria
AUTHOR
Houria
Ouled-Haddar
hrourou2002@gmail.com
2
1Laboratory of Molecular Toxicology, Faculty of Nature and life Sciences, University of Mohammed Seddik Benyahia - Jijel, 98 Ouled Aissa-Jijel 1800-Algeria, Algeria
AUTHOR
Mohamed
Sifour
sifourm@yahoo.fr
3
1Laboratory of Molecular Toxicology, Faculty of Nature and life Sciences, University of Mohammed Seddik Benyahia - Jijel, 98 Ouled Aissa-Jijel 1800-Algeria, Algeria
LEAD_AUTHOR
Cherifa
Beggah
beggahch@yahoo.fr
4
Department of Applied Microbiology and Food Sciences, Faculty of Nature and Life Sciences, University of Mohammed Seddik Benyahia- Jijel, 98 Ouled Aissa-Jijel 1800-Algeria, Algeria
AUTHOR
Farida
Benhamada
b.florida45@yahoo.fr
5
Department of Applied Microbiology and Food Sciences, Faculty of Nature and Life Sciences, University of Mohammed Seddik Benyahia- Jijel, 98 Ouled Aissa-Jijel 1800-Algeria, Algeria
AUTHOR
ORIGINAL_ARTICLE
Real-Time PCR: an Appropriate Approach to Confi rm ssDNA Generation from PCR Product in SELEX Process
Background: Aptamers are single stranded DNA (ssDNA) or RNA molecules. The potential of aptamers for binding to the different targets has made them be widely used as the preferred diagnostic and therapeutic tools. DNA aptamers present several advantages over the RNA oligonucleotides due to their higher stability, easier selection, and production. Selection of DNA aptamers which is facilitated through a systematic evolution of ligand by exponential enrichment (SELEX) method is much dependent on the successful conversion of double stranded DNA (dsDNA) to ssDNA.Objective: There are diff erent methods available for ssDNA generation. While visualization of ssDNA is limited to the gelbasedmethod, the method is not applicable in the initial rounds of SELEX due to more than 1015 diff erent sequences. Thisstudy was designed to evaluate the effi ciency of another technique for confi rming the ssDNA generation in comparison to the polyacrylamide electrophoresis (PAGE) analysis.Materials and Methods: Real-time PCR was employed in the present study for PCR amplifi cation of the initial library that was followed by enzymatic digestion of the dsDNA. Subsequently melting curve analysis was carried out to evaluate ssDNA generation from dsDNA. Moreover, PAGE analysis was performed and the results were compared with the melt curve analysis.Results: The melt curves, revealed dsDNA conversion to the ssDNA based on a signifi cant reduction of Tm from 73.8 to 41.5 °C. Applying PAGE analysis, it was not eff ectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was effi cient enough to confi rm ssDNA generation in accordance with the increasing the number of SELEX rounds.Conclusion: The present study has proven the applicability of the real-time PCR as a suitable confi rmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation.
https://www.ijbiotech.com/article_47066_8d1fb63047a34791b35eed25a30c0923.pdf
2017-04-01
143
148
DOI:10.15171/ijb.1550
Half- Renaturation
Melt Curve
PAGE
Real Time PCR
SELEX
Shirin
Kouhpayeh
shirin_ake@yahoo.com
1
Department of Immunology, Isfahan University of Medical Sciences, Isfahan, Iran.
AUTHOR
Zahra
Hejazi
zahrahejazi88@gmail.com
2
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 313, Iran
AUTHOR
Hossein
Khanahmad
h_khanahmad@med.mui.ac.ir
3
Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-communicable disease, Isfahan University of Medical Sciences, Isfahan, 313, Iran
AUTHOR
Abbas
Rezaei
kouhpayeh@resident.mui.ac.ir
4
Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 313, Iran
LEAD_AUTHOR