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Fulltext Recycling of superfine resolution agarose gel

Seng TY, Singh R, Faridah QZ, Tan SG, Alwee SS

Genet. Mol. Res., 2013;12(3):2360-7.
PMID: 23546970 DOI: 10.4238/2013.March.11.1

Genetic markers are now routinely used in a wide range of applications, from forensic DNA analysis to marker-assisted plant and animal breeding. The usual practice in such work is to extract the DNA, prime the markers of interest, and sift them out by electrically driving them through an appropriate matrix, usually a gel. The gels, made from polyacrylamide or agarose, are of high cost, limiting their greater applications in molecular marker work, especially in developing countries where such technology has great potential. Trials using superfine resolution (SFR) agarose for SSR marker screening showed that it is capable of resolving SSR loci and can be reused up to 14 times, thus greatly reducing the cost of each gel run. Furthermore, for certain applications, low concentrations of agarose sufficed and switching to lithium borate buffer, instead of the conventional Tris-borate-ethylenediaminetetraacetic acid buffer, will further save time and cost. The 2.5% gel was prepared following the Agarose SFR(TM) manual by adding 2.5 g agarose powder into 100 mL 1X lithium borate buffer in a 250-mL flask with rapid stirring. Two midigels (105 x 83 mm, 17 wells) or 4 minigels (50 x 83 mm, 8 wells), 4 mm thickness can be prepared from 100 mL gel solution. A total of 1680 PCR products amplified using 140 SSR markers from oil palm DNA samples were tested in this study using SFR recycled gel. As average, the gel can be recycled 8 times with good resolution, but can be recycled up to 14 times before the resolutions get blurred.

Matched MeSH terms: Sepharose/chemistry*
Fulltext Modified gel preparation for distinct DNA fragment analysis in agarose gel electrophoresis

Lee SV, Bahaman AR

Trop Biomed, 2010 Aug;27(2):351-4.
PMID: 20962737

Agarose gel electrophoresis is the standard method that is used to separate, identify, and purify DNA fragments. However, this method is time-consuming and capable of separating limited range of fragments. A new technique of gel preparation was developed to improve the DNA fragment analysis via electrophoresis.

Matched MeSH terms: Sepharose/chemistry
Solvent-impregnated agarose gel liquid phase microextraction of polycyclic aromatic hydrocarbons in water

Loh SH, Sanagi MM, Wan Ibrahim WA, Hasan MN

J Chromatogr A, 2013 Aug 9;1302:14-9.
PMID: 23809804 DOI: 10.1016/j.chroma.2013.06.010

A new microextraction procedure termed agarose gel liquid phase microextraction (AG-LPME) combined with gas chromatography-mass spectrometry (GC-MS) was developed for the determination of selected polycyclic aromatic hydrocarbons (PAHs) in water. The technique utilized an agarose gel disc impregnated with the acceptor phase (1-octanol). The extraction procedure was performed by allowing the solvent-impregnated agarose gel disc to tumble freely in the stirred sample solution. After extraction, the agarose gel disc was removed and subjected to centrifugation to disrupt its framework and to release the impregnated solvent, which was subsequently withdrawn and injected into the GC-MS for analysis. Under optimized extraction conditions, the new method offered high enrichment factors (89-177), trace level LODs (9-14ngL(-1)) and efficient extraction with good relative recoveries in the range of 93.3-108.2% for spiked drinking water samples. AG-LPME did not exhibit any problems related to solvent dissolution, and it provided high extraction efficiencies that were comparable to those of hollow fiber liquid phase microextraction (HF-LPME) and significantly higher than those of agarose film liquid phase microextraction (AF-LPME). This technique employed a microextraction format and utilized an environmentally compatible solvent holder that supported the green chemistry concept.

Matched MeSH terms: Sepharose/chemistry*
Characterisation of a Ferrous Agarose Xylenol (FAX) gel for radiotherapy dose measurement

Leong LH, Kandaiya S, Seng NB

Australas Phys Eng Sci Med, 2007 Jun;30(2):135-40.
PMID: 17682403

The oxidation of ferrous to ferric ions due to ionizing radiation has been used for chemical dosimetry since 1927. The introduction of metal indicator dye xylenol orange (XO) sensitises the measurement of ferric ion yield. A ferrous sulphate- agarose- xylenol orange (FAX) gel was prepared and the gel then exposed to dose ranging from 0.2 to 10 Gy using various high energy photon and electron beams from a linear accelerator. Some general characteristics of FAX such as energy dependence, optical density (OD)-dose relationship, reproducibility and auto-oxidation of ferrous ions were analysed. The radiation yield G of the gel was calculated for gels prepared in oxygen and in air and the values were 46.3 +/- 2.1 and 40.9 +/- 1.4 Fe3+ per 100 eV for photons respectively. However for stock gel which was kept for 5 days pre-irradiation the G value decreased to 36.6 +/- 1.1. The gel shows linearity in OD-dose relationship, energy independence and reproducibility over the dose range investigated. Auto-oxidation of ferrous ions resulted in optical density changes of less than 1.5% per day.

Matched MeSH terms: Sepharose/chemistry*
Fulltext DNA horizontal polyacrylmide gel electrophoresis

Elsie Yee, Y. S., Zainal Zahari, AHMAD ISMAIL, YAP, C.K., TAN, S. G

Buletin Persatuan Genetik Malaysia, 2010;16(1):19-22.
MyJurnal

Electrophoresis is a crucial step for the studies of proteins, allozymes, DNAs and RNAs. Two commonly used electrophoresis systems are agarose gel and polyacrylmide gel. Agarose gel is frequently used for DNAs and RNAs studies whereas polyacrylmide gel is widely used for the studies of other macromolecules such as proteins, allozymes (isozymes), DNAs and RNAs. The banding patterns of the gels, rather than the numbers of bands appearing on the gels are important for scoring in fingerprinting, footprinting and in population genetic studies.

Matched MeSH terms: Sepharose
Development of the compact UV illuminator with single UV lamp

Lee DJ, Kim SY, Kim JD, Kim YS, Song HJ, Park CY

Sains Malaysiana, 2015;44:1693-1699.

This paper presents a low-cost method of constructing the compact UV illuminator, which is considered as an important
component of a gel documentation system. The procedure involves using a smallest-possible UV lamp and a motor which
moves the UV lamp in the UV illuminator instead of conventional 4 UV lamps. A comparative analysis of images produced
by using the commercial gel documentation system and our prototype was carried out. These comparisons were done
in real DNA gel as well as a reference plate made of quantum dot. The plate was composed of the chambers filled with
various densities of the quantum dot instead of the Agarose gel containing the ETBR in order to increase the accuracy of
comparison and the convenience of experiments. Despite the use of only 1 UV lamp, the proposed system demonstrated
a similar imaging performance compared with the conventional gel documentation system equipped with 4 UV lamps,
resulting in the great reduction of the system cost.

Matched MeSH terms: Sepharose
Agarose- and alginate-based biopolymers for sample preparation: Excellent green extraction tools for this century

Sanagi MM, Loh SH, Wan Ibrahim WN, Pourmand N, Salisu A, Wan Ibrahim WA, et al.

J Sep Sci, 2016 Mar;39(6):1152-9.
PMID: 27027592 DOI: 10.1002/jssc.201501207

Recently, there has been considerable interest in the use of miniaturized sample preparation techniques before the chromatographic monitoring of the analytes in unknown complex compositions. The use of biopolymer-based sorbents in solid-phase microextraction techniques has achieved a good reputation. A great variety of polysaccharides can be extracted from marine plants or microorganisms. Seaweeds are the major sources of polysaccharides such as alginate, agar, agarose, as well as carrageenans. Agarose and alginate (green biopolymers) have been manipulated for different microextraction approaches. The present review is focused on the classification of biopolymer and their applications in multidisciplinary research. Besides, efforts have been made to discuss the state-of-the-art of the new microextraction techniques that utilize commercial biopolymer interfaces such as agarose in liquid-phase microextraction and solid-phase microextraction.

Matched MeSH terms: Sepharose
Fulltext T1 and T2 Characteristics of Agarose Gel Phantom with different Gadolinium Oxide Concentration as Relaxation Modifier

Azhar, N. A. A., Tee, H. S., Yee, Y. Y., Awang, M. N. A., Abdul Manan, H., Yusoff, A. N.

Physics and Technology in Medicine, 2020;1(1):27-37.
MyJurnal

Many studies have been carried out to produce magnetic resonance imaging (MRI) phantoms as alternative to water phantom. Among the important properties of a phantom are the T1 and T2 relaxation times. The objective of this study is to investigate the T1 and T2 characteristics of the agarose gel phantoms with different relaxation modifier (gadolinium (III) oxide, Gd2O3) concentrations or [Gd2O3]. Six agarose gel phantoms were prepared with different [Gd2O3]. The T1 (fixed echo time (TE) and different repetition time (TR)) and T2 (fixed TR and different TE) measurements on all phantoms were conducted using the 3-T MRI system via spin echo (SE) and turbo spin echo (TSE) sequences, respectively. The signal-to-noise ratio (SNR) of all phantoms was calculated using Image-J software by implementing the region of interest (ROI) analysis. The SNR against TR and SNR against TE curves were fitted to the exponential equations for saturation, T1 and T2 determination. For every phantom, T1 curve demonstrated that the SNR increased exponentially with increasing TR, while T2 curves showed that the SNR decreased exponentially with increasing TE. Gd2O3 was found to successfully act as the relaxation modifier for the T1 but not the T2 curves. The T1 curve started to show saturated SNR (SNRo) and increasing SNRo for TR > 1000 ms and [Gd2O3] = 0.005 g/ml or higher. These behaviours are explained based on the dipole-dipole interaction that increases in phantoms with higher [Gd2O3], thus shortening the T1 relaxation. However, a systematic change in the T2 parameters with increasing [Gd2O3] was not observed. While Gd2O3 has significant effects on T1 relaxation parameters, the T2 relaxation parameters were minimally affected. With a shorter T1, the Gd2O3 added agarose gel can potentially be used as test phantom in fast imaging sequence, e.g. gradient echo pulse sequences.

Matched MeSH terms: Sepharose
Fulltext Effects of Reduced Gadolinium-Based Contrast Agents (GBCA) Volumes on MRI Image Quality: An Experimental Phantom Study

Ismail, N., Bashah, F. A. A., Zakaria, F.

Physics and Technology in Medicine, 2020;1(2):32-39.
MyJurnal

Many recent studies focused on the patient’s safety from the administration of gadolinium-based contrast agents (GBCAs), their concentration, the dose of administration and their effects on the image quality. The present study was aimed at evaluating the effects of reduced GBCAs (gadobutrol and gadoterate meglumine) volume on the image quality by using phantoms. Eight (8) human brain mimicking phantom made of nickel chloride (NiCl2) doped agarose gel were added with 0.00500 ml (100% volume), 0.00350 ml (75% volume), 0.00250 ml (50% volume) and 0.00125 ml (25% volume) of gadobutrol, 0.0100 ml (100% volume), 0.0075 ml (75% volume), 0.0050 ml (50% volume) and 0.0025 ml (25% volume) of gadoterate meglumine. The phantoms were scanned using a 1.5-T and a 3 T-MRI system. Signal-to-noise ratio (SNR) and the contrast agents enhancement were evaluated quantitatively and qualitatively. The 50% volume of gadobutrol and gadoterate meglumine at 3 T showed greater enhancement when compared with 50% and 100% volumes of gadobutrol and gadoterate meglumine at 1.5 T. It can be concluded that the volume of gadobutrol and gadoterate meglumine contrast agents can be reduced when using a higher field system

Matched MeSH terms: Sepharose
Fulltext Utilization of an Ionic Liquid/Urea Mixture as a Physical Coupling Agent for Agarose/Talc Composite Films

Shamsuri AA, Daik R

Materials (Basel), 2013 Feb 22;6(2):682-698.
PMID: 28809334 DOI: 10.3390/ma6020682

An ionic liquid, 1-n-butyl-3-methylimidazolium chloride (BmimCl) was blended with urea at 1:1 mole ratio to create a BmimCl/Urea mixture. The agarose/talc composite films containing the BmimCl/Urea mixture were then acquired through a gelation method. The weight ratio of agarose and talc was fixed at 4:1, while the content of BmimCl/Urea was varied from 0 to 10 wt % relative to the overall weight of the composite films. The tensile stress and modulus results showed the optimum BmimCl/Urea content in the composite film lies at 8 wt %. The talc particles are embedded in the agarose matrix and there are no pullouts for the composite films containing BmimCl/Urea as demonstrated by SEM micrographs. The addition of BmimCl/Urea increased the glass transition temperature of the composite films, however, the thermal decomposition temperature decreased drastically. FTIR and FT-Raman spectra indicated the existence of interaction between agarose and talc, which improves their interfacial adhesion. As a conclusion, a BmimCl/Urea mixture can be utilized as a coupling agent for agarose/talc composite films.

Matched MeSH terms: Sepharose
A green solvent holder in electro-mediated microextraction for the extraction of phenols in water

Chong YT, Mohd Ariffin M, Mohd Tahir N, Loh SH

Talanta, 2018 Jan 01;176:558-564.
PMID: 28917790 DOI: 10.1016/j.talanta.2017.08.068

Electro-mediated microextraction (EMM) combined with micro-high performance liquid chromatography-ultraviolet detection was successfully developed for the determination of selected phenols, namely 4-chlorophenol (4CP), 2-nitrophenol (2NP) and 2,4-dichlorophenols (2,4 DCP) in water. A solvent-impregnated agarose gel disc was utilized as a solvent holder in this study. Under optimum extraction conditions, the method showed good linearity in the range of 0.1-250µgL-1, 0.3-250µgL-1and 0.2-500µgL-1for 4CP, 2NP and 2,4 DCP, respectively with correlation coefficients of ≥ 0.9975, ultra-trace LODs (0.03-0.1µgL-1) and satisfactory relative recovery average (85.0-114.1%) for the analysis of selected phenols. The proposed method was rapid and eco-friendly as the solvent holder was constructed using minute amounts of extraction solvent immobilized within the biodegradable agarose gel disc. A comparative microextraction technique termed solvent-impregnated agarose gel liquid phase microextraction (AG-LPME) was re-optimized and validated for the extraction of phenols in water. The method offered good linearity, ultra-trace LODs ranging 0.1-0.5µgL-1and satisfactory average of relative recovery (86.1-114.1%). The EMM was superior in terms of sensitivity and time-effectiveness compared to AG-LPME. Both techniques combine extraction and pre-concentration in mini-scaled approaches using an eco-friendly solvent holder that fulfil the green chemistry concept.

Matched MeSH terms: Sepharose
Multi-walled carbon nanotube-impregnated agarose film microextraction of polycyclic aromatic hydrocarbons in green tea beverage

Loh SH, Sanagi MM, Wan Ibrahim WA, Hasan MN

Talanta, 2013 Mar 15;106:200-5.
PMID: 23598117 DOI: 10.1016/j.talanta.2012.12.032

A new microextraction procedure termed multi-walled carbon nanotube-impregnated agarose film microextraction (MWCNT-AFME) has been developed. The method utilized multi-walled carbon nanotubes (MWCNTs) immobilized in agarose film to serve as adsorbent in solid phase microextraction (SPME). The film was prepared by mixing the MWCNTs in agarose solution and drying the mixture in oven. Extraction of selected polycyclic aromatic hydrocarbons was performed by inserting a needle through circular MWCNT-impregnated agarose films (5 mm diameter) and the assembly was dipped into an agitated sample solution prior to micro high performance liquid chromatography-ultraviolet analysis. Back extraction was then performed using ultrasonication of the films in 100 μL of solvent. The film was discarded after single use, thus avoiding any analyte carry-over effect. Due to the mesoporous nature of the agarose film, the MWCNTs were immobilized easily within the film and thus allowing for close contact between adsorbent and analytes. Under the optimized extraction conditions, the technique achieved trace LODs in the range of 0.1 to 50 ng L(-1) for the targeted analytes, namely fluoranthene, phenanthrene and benzo[a]pyrene. The method was successfully applied to the analysis of spiked green tea beverage samples with good relative recoveries in the range of 91.1 to 107.2%. The results supported the feasibility of agarose to serve as adsorbent holder in SPME which then minimizes the consumption of chemicals and disposal cost of organic wastes.

Matched MeSH terms: Sepharose/chemistry*
Agarose-chitosan-C18 film micro-solid phase extraction combined with high performance liquid chromatography for the determination of phenanthrene and pyrene in chrysanthemum tea samples

Ng NT, Sanagi MM, Wan Ibrahim WN, Wan Ibrahim WA

Food Chem, 2017 May 01;222:28-34.
PMID: 28041555 DOI: 10.1016/j.foodchem.2016.11.147

Agarose-chitosan-immobilized octadecylsilyl-silica (C18) film micro-solid phase extraction (μSPE) was developed and applied for the determination of phenanthrene (PHE) and pyrene (PYR) in chrysanthemum tea samples using high performance liquid chromatography-ultraviolet detection (HPLC-UV). The film of blended agarose and chitosan allows good dispersion of C18, prevents the leaching of C18 during application and enhances the film mechanical stability. Important μSPE parameters were optimized including amount of sorbent loading, extraction time, desorption solvent and desorption time. The matrix match calibration curves showed good linearity (r⩾0.994) over a concentration range of 1-500ppb. Under the optimized conditions, the proposed method showed good limits of detection (0.549-0.673ppb), good analyte recoveries (100.8-105.99%) and good reproducibilities (RSDs⩽13.53%, n=3) with preconcentration factors of 4 and 72 for PHE and PYR, respectively.

Matched MeSH terms: Sepharose/chemistry
Size-selective purification of hepatitis B virus-like particle in flow-through chromatography: Types of ion exchange adsorbent and grafted polymer architecture

Ng HW, Lee MFX, Chua GK, Gan BK, Tan WS, Ooi CW, et al.

J Sep Sci, 2018 May;41(10):2119-2129.
PMID: 29427396 DOI: 10.1002/jssc.201700823

Hepatitis B virus-like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow-through chromatography mode. The virus-like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus-like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch-chain length) of the grafted polymer. The branch-chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 μm) had an approximately twofold increase in the flow-through recovery, as compared to the smaller adsorbent (30 μm). Generally, polymer-grafted adsorbents improved the exclusion of the virus-like particles. Overall, the middle branch-chain length polymer grafted on larger adsorbent showed optimal performance at 92% flow-through recovery with a purification factor of 1.53. A comparative study between the adsorbent with dextran grafts and the polymer-grafted adsorbent showed that a better exclusion of virus-like particles was achieved with the absorbent grafted with inert polymer. The grafted polymer was also shown to reduce strong interaction between binding sites and virus-like particles, which preserved the particles' structure.

Matched MeSH terms: Sepharose/chemistry
MOLECULAR CLONING AND BIOCHEMICAL CHARACTERIZATION OF GALACTOSE-1-PHOSPHATE URIDYLYLTRANSFERASE FROM GRACILARIA CHANGII (RHODOPHYTA)(1)

Siow RS, Teo SS, Ho WY, Shukor MY, Phang SM, Ho CL

J Phycol, 2012 Feb;48(1):155-62.
PMID: 27009660 DOI: 10.1111/j.1529-8817.2011.01105.x

Galactose-1-phosphate uridylyltransferase (GALT) catalyzes the reversible conversion of glucose-1-phosphate and UDP-galactose to galactose-1-phosphate and UDP-glucose. This enzyme is also responsible for one of the biochemical steps that produce the precursors of agar and agarose. In this study, we report the molecular cloning and sequence analyses of a cDNA encoding GALT, from Gracilaria changii (B. M. Xia et I. A. Abbott) I. A. Abbott, J. Zhang et B. M. Xia, which constitutes a genus of seaweeds that supply more than 60% of the world's agar and agarose. We have subcloned this cDNA into a bacterial expression cloning vector and characterized the enzyme activities of its recombinant proteins in vitro. The GcGALT gene was shown to be up-regulated by salinity stresses. The abundance of transcripts encoding GcGALT was the highest in G. changii, followed by Gracilaria edulis and Gracilaria salicornia in a descending order, corresponding to their respective agar contents. Our findings indicated that GALT could be one of the components that determines the agar yield in Gracilaria species.

Matched MeSH terms: Sepharose
Fulltext Dynamic Hepatocellular Carcinoma Model Within a Liver Phantom for Multimodality Imaging

Ahmad MS, Suardi N, Shukri A, Nik Ab Razak NNA, Oglat AA, Makhamrah O, et al.

Eur J Radiol Open, 2020;7:100257.
PMID: 32944594 DOI: 10.1016/j.ejro.2020.100257

Introduction: Hepatocellular carcinoma (HCC) is one of the most common cancer in the world, and the effectiveness of its treatment lies in its detection in its early stages. The aim of this study is to mimic HCC dynamically through a liver phantom and apply it in multimodality medical imaging techniques including magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound.
Methods and materials: The phantom is fabricated with two main parts, liver parenchyma and HCC inserts. The liver parenchyma was fabricated by adding 2.5 wt% of agarose powder combined with 2.6 wt% of wax powder while the basic material for the HCC samples was made from polyurethane solution combined with 5 wt% glycerol. Three HCC samples were inserted into the parenchyma by using three cylinders implanted inside the liver parenchyma. An automatic injector is attached to the input side of the cylinders and a suction device connected to the output side of the cylinders. After the phantom was prepared, the contrast materials were injected into the phantom and imaged using MRI, CT, and ultrasound.
Results: Both HCC samples and liver parenchyma were clearly distinguished using the three imaging modalities: MRI, CT, and ultrasound. Doppler ultrasound was also applied through the HCC samples and the flow pattern was observed through the samples.
Conclusion: A multimodal dynamic liver phantom, with HCC tumor models have been fabricated. This phantom helps to improve and develop different methods for detecting HCC in its early stages.

Matched MeSH terms: Sepharose
Fulltext A modified method for genomic DNA extraction from the fish intestinal microflora

Han Z, Sun J, Lv A, Sung Y, Sun X, Shi H, et al.

AMB Express, 2018 Apr 02;8(1):52.
PMID: 29610998 DOI: 10.1186/s13568-018-0578-3

A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.

Matched MeSH terms: Sepharose
Neonatal buccal cell collection: a non-invasive strategy to provide a reliable source of DNA for high resolution melting analysis

Cheung, Tian Pei, Rostenberghe, Hans Van, Narazah Mohd Yusoff, Noraida Ramli, Nor Rosidah Ibrahim, Nishio, Hisahide, et al.

Malaysian Journal of Paediatrics and Child Health, 2019;25(2):14-22.
MyJurnal

Background: The low yield and quality of buccal-derived genomic DNA have reduced its applicability in various genetic research. The aim of this study was to assess the quantity, purity and genotyping efficiency of genomic DNA isolated from neonatal buccal swabs. Methods: Paired buccal swabs and whole blood samples were collected from 60 neonates with the mean age 5 days (SD=1.57). The genomic DNA quantity and purity were measured by using Infinite® 200 PRO NanoQuant reader and agarose gel electrophoresis. High-resolution melting (HRM) analysis was used to analyse the sequence variants present in uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1 c.211G>A) and nuclear receptor subfamily 1, group I, member 3 (NR1I3 IVS8+116T>G) genes. Results: Buccal swabs provided lower mean genomic DNA concentration (18.78 ± 8.39 ng/μl versus 40.02 ± 13.03 ng/μl), yield (2.63 ± 1.17 μgversus8.00 ± 2.61 μg). The purity of buccal samples however were inconsistent with 16 samples (26.7%) having A260/280 ratios below 1.8 which indicated protein contamination. Genomic DNA purity for all blood samples were within the ideal range with average absorbance ratios of 1.8−2.0. However, all buccal genomic DNA demonstrated 100% genotype call rates for all variants. A complete genotype concordance was also observed between paired genomic DNA samples. Conclusion: Despite related to a reduced quantity and purity, neonatal buccal genomic DNA could generate reliable HRM genotyping results. Therefore, buccal swab collection is a promising alternative to the invasive blood sampling to provide genomic DNA for genetic analysis involving paediatric population.

Matched MeSH terms: Sepharose
First Report of Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum on Cucumber in Malaysia

Nazerian E, Sijam K, Zainal Abidin MA, Vadamalai G

Plant Dis, 2011 Nov;95(11):1474.
PMID: 30731752 DOI: 10.1094/PDIS-10-10-0754

Cucumber (Cucumis sativus L.) is one of the most important vegetable fruits in Malaysia. Cucumber is principally grown in the states of Johor, Kelantan, and Perak. The broad host range Enterobacteriaceae pathogen, Pectobacterium carotovorum, can cause soft rot on stems or cucumber fruit. In Malaysia, cucumber is produced in a warm, humid climate, thus the plant is susceptible to attack by P. carotovorum at any time during production. In 2010, cucumber samples with wilted and chlorotic leaves, water-soaked lesions, and collapsed fruits were found in multiple fields. Small pieces of infected stems and fruit were immersed in 5 ml of saline solution (0.85% NaCl) for 20 min and then 50 μl of this suspension was spread onto nutrient agar (NA) and incubated at 27°C for 24 h. White-to-pale gray colonies with irregular margins were selected for analysis. For pathogenicity tests, cucumber fruits were surface sterilized by ethyl alcohol 70%, washed with sterilized distilled water, cut into small pieces, and inoculated with 20 μl of 108 CFU/ml suspensions of five representative strains. Cucumber plants were grown for 3 weeks in sterilized soil and their stems were inoculated with 20 μl of 108 CFU/ml of bacterial suspension. Inoculated samples and control (noninoculated) plants were placed in a growth chamber with 80 to 90% relative humidity at 27°C. Symptoms occurred on fruit slices and stems after 1 to 3 days and appeared the same as naturally infected samples, but the control samples remained healthy. Koch's postulates were fulfilled with the reisolation of cultures with the same characteristics as described earlier. Hypersensitivity reaction (HR) assays were done by infiltrating 108 CFU/ml of bacterial suspension into tobacco leaf epidermis and HR developed. All strains were subjected to biochemical and morphological assays, as well as molecular assessment. The strains were gram negative, facultative anaerobes, rod shaped, able to macerate potato slices and growth at 37°C; catalase positive; oxidase and phosphatase negative; able to degrade pectate; sensitive to erythromycin; negative for utilization of α-methyl glycoside, indole production, and reduction of sugars from sucrose; acid production from arabitol, sorbitol, and utilization of citrate were negative, but positive for raffinose and melibiose utilization. PCR amplification of the pel gene by Y1 and Y2 primers produced a 434-bp fragment on agarose gel 1% (1). Amplification of intergenic transcribed spacer region by G1 and L1 primers gave two main bands at approximately 535 and 580 bp on agarose gel 1.5%. The ITS-PCR products were digested with RsaI restriction enzyme (3). On the basis of biochemical and morphological characteristics, PCR-based pel gene and characterization of the ITS region, and digestion of the ITS-PCR products with RsaI restriction enzyme, all isolates were identified as P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of soft rot caused by P. carotovorum subsp. carotovorum on cucumber from Malaysia. References: (1) A. Darraas et al. Appl. Environ. Microbiol. 60:1437, 1994. (2) N. W Schaad et al. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society Press, St. Paul, 2001. (3) I. K. Toth et al. Appl. Environ. Microbiol. 67:4070, 2001.

Matched MeSH terms: Sepharose
Fulltext Isolation, Purification, and Characterization of Heparinase from Streptomyces variabilis MTCC 12266

Singh V, Haque S, Kumari V, El-Enshasy HA, Mishra BN, Somvanshi P, et al.

Sci Rep, 2019 04 24;9(1):6482.
PMID: 31019210 DOI: 10.1038/s41598-019-42740-7

Arterial/venous thrombosis is the major cardiovascular disorder accountable for substantial mortality; and the current demand for antithrombotic agents is extensive. Heparinases depolymerize unfractionated heparin (UFH) for the production of low molecular-weight heparins (LMWHs; used as anticoagulants against thrombosis). A microbial strain of Streptomyces sp. showing antithrombotic activity was isolated from the soil sample collected from north India. The strain was characterized by using 16S rRNA homology technique and identified as Streptomyces variabilis MTCC 12266 capable of producing heparinase enzyme. This is the very first communication reporting Streptomyces genus as the producer of heparinase. It was observed that the production of intracellular heparinase was [63.8 U/mg protein (specific activity)] 1.58 folds higher compared to extracellular heparinase [40.28 U/mg protein]. DEAE-Sephadex A-50 column followed by Sepharose-6B column purification of the crude protein resulted 19.18 folds purified heparinase. SDS-PAGE analysis of heparinase resulted an estimated molecular-weight of 42 kDa. It was also found that intracellular heparinase has the ability to depolymerize heparin to generate LMWHs. Further studies related to the mechanistic action, structural details, and genomics involved in heparinase production from Streptomyces variabilis are warranted for large scale production/purification optimization of heparinase for antithrombotic applications.

Matched MeSH terms: Sepharose

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