MOSP - Macao Scientific Publishers

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Bioinformatic tools and guideline for PCR primer design

Kamel A. Abd-Elsalam

Abstract: Bioinformatics has become an essential tool not only for basic research but also for applied research in biotechnology and biomedical sciences. Optimal primer sequence and appropriate primer concentration are essential for maximal specificity and efficiency of PCR. A poorly designed primer can result in little or no product due to non-specific amplification and/or primer-dimer formation, which can become competitive enough to suppress product formation. There are several online tools devoted to serving molecular biologist design effective PCR primers. This review intends to provide a guide to choosing the most efficient way to design a new specific-primer by applying current publicly available links and Web services. Also, the purpose here is to provide general recommendations for the design and use of PCR primers.[...] Read More.

Keywords: Bio-computing, primer design, web-based resources.

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A home made kit for plasmid DNA mini-preparation

Simeon Oloni KOTCHONI , Emma Wanjiru GACHOMO, Eriola BETIKU, and Olusola Olusoji SHONUKAN

Abstract: Many methods have been used to isolate plasmid DNA, but some of them are time consuming especially when extracting a large number of samples. Here, we developed a rapid protocol for plasmid DNA extraction based on the alkaline lysis method of plasmid preparation (extraction at pH 8.0). Using this new method, a good plasmid preparation can be made in approximately one hour. The plasmids are suitable for any subsequent molecular applications in the laboratory. By applying the recommendations to avoid contaminations and to maximize the plasmid yield and quality during extraction, this protocol could be a valuable reference especially when analyzing a large number of samples.[...] Read More.

Keywords: Plasmid extraction, PCR, restriction enzymes, sequencing, contamination.

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Genetic comparisons of Egyptian date palm cultivars (Phoenix dactylifera L.) by RAPD-PCR

Said Saad Soliman, Bahy Ahmed Ali*, Mohamed Morsy Mohamed Ahmed

Abstract: Random amplified polymorphic DNA technique was used to compare genetic material from four females date palm and four unknown male trees of Egyptian date palm. The genetic similarity between the four females date palm (Zaghloul, Amhat, Samany and Siwi) ranged from 87.5 to 98.9%. The banding profiles obtained suggested that both males 3 and 4 are genetically related to the four female cultivars.[...] Read More.

Keywords: Date palm, cultivars, RAPD-PCR, genetic similarity.

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A capillary electrophoretic method for isolation and characterization of grape xylem proteins

Ashok K. Jain* and Sheikh M. Basha

Abstract: European (Vitis vinifera) and American (Vitis labrusca) grape species succumb to a bacterial disease known as Pierce’s Disease (PD). In contrast, muscadine grape genotypes (Vitis rotundifolia) are tolerant/resistant to PD. This is due to the unique biochemical composition of muscadine xylem. However, because of low protein concentration, conventional methods such as low-pressure chromatography and PAGE are unsuitable for grape xylem protein characterization. In addition, these procedures are tedious, time-consuming and require large amount of sample. This study reports a procedure for isolating and separating proteins from muscadine and bunch grape xylem tissue. The procedure consists of separation of xylem from cortex and phloem, removal of pigments and other gummy substances from xylem with ethanol: ethylacetate (2:1) and subsequent Capillary Electrophoretic (CE) analysis of xylem protein extracts to achieve desired resolution. Number of peaks, peak height and areas, retention time and baseline position were used to compare resolution and study the effect of sample and separation buffer. Xylem tissue proteins extracted with 0.05% sodium borate buffer (pH 8.3) and subjected to CE using 1.2% sodium borate (pH 8.3) as a separation buffer were found to yield most satisfactory resolution of grape xylem proteins. The data obtained by CE were consistent and reproducible, and hence, is well suited to obtain excellent resolution of xylem tissue protein for identifying differences in protein composition among the grape genotypes.[...] Read More.

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Functional and comparative analysis of expressed sequences from Diuraphis noxia infested wheat obtained utilizing the conserved Nucleotide Binding Site

Lynelle Lacock, Chantal van Niekerk, Shilo Loots, Franco du Preez and Anna-Maria Botha*

Abstract: Russian wheat aphid (Diuraphis noxia, Morvilko; RWA) is a major pest on wheat, barley and other triticale in South Africa. Infestation by the RWA results in altered protein expression patterns, which is manifested as differential expression of gene sequences. In the present study, Russian wheat aphid resistant (Tugela DN, Tugela*5/SA2199, Tugela*5/SA463, PI 137739, PI 262660, and PI 294994) and susceptible triticale (Tugela) were infested and cDNA synthesized. A PCR based approach was utilized to amplify the nucleotide binding site conserved region to obtain expressed sequence tags (ESTs) with homology to resistance gene analogs (RGAs). The approach proved highly feasible when the isolation of RGAs is the main objective, since 18% of all obtained ESTs showed significant hits with known RGAs, when translated into their corresponding amino acid sequences and searched against the nonredundant GenBank protein database using the BLASTX algorithm.[...] Read More.

Keywords: Resistance gene analogs, degenerate PCR, nucleotide-binding site-leucine rich repeat resistance genes, Aegilops tauschii.

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Acinetobacter: Environmental and biotechnological applications

Desouky Abdel-El-Haleem

Abstract: Among microbial communities involved in different ecosystems such as soil, freshwater, wastewater and solid wastes, several strains belonging to the genus of Acinetobacter have been attracting growing interest from medical, environmental and a biotechnological point of view. Bacteria of this genus are known to be involved in biodegradation, leaching and removal of several organic and inorganic man-made hazardous wastes. It is also well known that some of Acinetobacter strains produce important bioproducts. This review summarizes the usefulness and environmental applications of Acinetobacter strains.[...] Read More.

Keywords: Acinetobacter, biodegradation, xenobiotic, oil, heavy metals, bioproducts, lipases, polysaccharides.

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Production of alkaline protease by Teredinobacter turnirae cells immobilized in Ca-alginate beads

Usama Beshay

Abstract: The conditions for immobilizing the new alkaline protease-producing bacteria strain Teredinobacter turnirae by entrapment in calcium alginate gel were investigated. The influence of alginate concentration (20, 25 and 30 g/l) and initial cell loading (ICL) on enzyme production were studied. The production of alkaline protease improved significantly with increasing alginate concentration and reached a maximum enzyme yield of 8000 U/ml at 25 g/l alginate concentration. This was about 176.8% higher than that obtained by free cells (2890 U/ml). The immobilized cells produced alkaline protease consistently over 5 repeated cycles and reached a maximal value of 9000 U/ml on the third cycle. This was 311.4% (3.11-fold) as compared with the control (free cells). Simple mass balance analysis was applied to describe the growth and the protease production behaviour of both fractions the cells in free form and the entrapped in Ca-alginate beads. Scanning electron microscope studies indicated the internal distribution pattern of the cells encapsulated in Ca-alginate beads. The results presented in this paper show the potential for using immobilized T. turnirae cells in Ca-alginate for the production of a novel alkaline protease.[...] Read More.

Keywords: Alkaline protease, Ca-alginate, immobilization, Teredinobacter turnirae, repeated batch.

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Molecular phylogeny of Fusarium species by AFLP fingerprint

Mohmed A. Abdel-Satar, Mohmed. S. Khalil, I. N. Mohmed, Kamel A. Abd-Elsalam,*, and Joseph A. Verreet

Abstract: The high- resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships within and between natural populations of five Fusarium spp. AFLP templates were prepared by the digestion of Fusarium DNA with EcoRI and MseI restriction endonucleases and subsequent ligation of corresponding site-specific adapters. An average of 44 loci was assayed simultaneously with each primer pair and DNA markers in the range 100 to 500 bp were considered for analysis. A total of 80 AFLP polymorphic markers were obtained using four primer combinations, with an average of 20 polymorphic markers observed per primer pair. UPGMA analyses indicated 5 distinct clusters at the phenon line of 30% on the genetic similarity scale corresponding to the 5 taxa. The similarity percent of each group oscillated between 87 and 97%. The phenetic dendrogram generated by UPGMA as well as principal coordinate analysis (PCA) grouped all of the Fusarium spp. isolates into five major clusters. No clear trend was detected between clustering in the AFLP dendrogram and geographic origin, host genotype of the tested isolates with a few exceptions. The results of the present study provide evidence of the high discriminatory power of AFLP analysis, suggesting the possible applicability of this method to the molecular characterization of Fusarium.[...] Read More.

Keywords: AFLP, Fusarium, molecular phylogeny, selective amplification.

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Relationship between genetic similarity and some productive traits in local chicken strains

Bahy Ahmed Ali*, Mohamed Morsy Mohamed Ahmed, Osama Mahmoud Aly

Abstract: Random amplified polymorphic DNA (RAPD) technique was applied to detect genetic similarity between five local chicken strains that have been selected for eggs and meat production in Egypt. Based on six oligonucleotide primers, the genetic similarity between the egg -producing strains (Anshas, Silver Montazah and Mandarah) ranged from 72.4 to 85.4%. While the genetic similarity between the two chicken strains selected for meat production (Baheij and El-Salam) is 86.9%.[...] Read More.

Keywords: Chicken, local strains, RAPD-PCR, genetic similarity.

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Variations in fatty acid proportions during desiccation of Telfairia occidentalis seeds harvested at physiological and agronomic maturity

Nkang A*, Omokaro D, Egbe A and Amanke, G

Abstract: The effect of desiccation on lipid content, fatty acid composition and the antioxidative enzymic capacity was investigated in seeds of Telfairia occidentalis, harvested at physiological and agronomic maturity. Seeds were dried at 5 and 28 oC, environments that induced different drying and metabolic rates. Desiccation of seeds was associated with decreased antioxidative enzymic capacity (of peroxidase and polyphenoloxidase), and thus increased likelihood of free radical attack and decreased viability (germinability). Agronomically mature seeds contained predominantly saturated fatty acids (tridecanoic), with very low levels of the major fatty acids of edible oilseeds (palmitic, stearic or the unsaturated C18 fatty acids). There was increased accumulation of the mono-unsaturated (oleic) and polyunsaturated (linoleic) fatty acids when seeds were dried at 28 oC and moisture contents have reduced to about 42 % or lower. In contrast, seeds dried at 5 oC maintained high levels of saturated fatty acids and lower levels of monounsaturated or polyunsaturated fatty acids. Results suggest the need to develop different post -harvest protocols for seed storage, and for processing T. occidentalis to ‘improve’ the seed fatty acid profile as an oilseed for human and animal food.[...] Read More.

Keywords: Agronomic maturity, desiccation, fatty acid, lipid peroxidation, oilseed.

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