Volume 3 Special Issue 1 2009
Focus on Bioinformatics
How to reference: Kalendar R, Lee D, Schulman AH (2009) FastPCR Software for PCR Primer and Probe Design and Repeat Search. In: Mansour A (Ed) Focus on Bioinformatics. Genes, Genomes and Genomics 3 (Special Issue 1), 1-14
Ahmed Mansour Mohamed Mansour Alzohairy
Genetics Department, Faculty of Agriculture, Zagazig University, Egypt
CONTENTS AND ABSTRACTS
Ruslan Kalendar (Finland), David Lee (UK), Alan H. Schulman (Finland) FastPCR Software for PCR Primer and Probe Design and Repeat Search (pp 1-14)
Invited Review: Reproducible and target-specific polymerase chain reaction (PCR) amplification relies on several interrelated factors of which primer design is central. Here, we describe new free bioinformatics software, the FastPCR which was developed, and continues to be updated, based on detailed experimental studies of PCR efficiency for the optimal design of primers and probe sequences and for repeat searching. This software forms an environment of integrated tools, which provides comprehensive facilities for designing primers for most PCR applications including multiplex and self-reporting fluorescent systems. FastPCR consists of a data editor, build commands for probe and primer design, and automation tools. The software selects the best primers with the widest range of melting temperatures, which allows designing qualified primers for all PCR tasks. The “in silico” PCR primer or probe searching includes comprehensive individual primers and primer pair analysis tests. FastPCR utilizes combinations of normal and degenerate primers for all tools. The melting temperature calculation is based on nearest neighbour thermodynamic parameters starting with multiple nucleic acid or protein sequences. It performs efficient and complete detection of various repeat types with visual display. FastPCR is able to perform repeat searches for a single sequence or for comparisons of two sequences. The program includes various bioinformatics tools for analysis of sequences with GC or AT skew, GC content, and purine-pyrimidine skew, and considers linguistic sequence complexity. It can generate random DNA sequence, make restriction analysis, and supports the clustering of sequences and consensus sequence generation, as well as sequence similarity and conservancy analyses.
Zohra Khalis, Jean-Paul Comet, Adrien Richard, Gilles Bernot (France) The SMBioNet Method for Discovering Models of Gene Regulatory Networks (pp 15-22)
Invited Mini-Review: To study gene regulatory networks, we work on an iterative approach that permits us via formal modelling to elaborate models in silico and to validate them in vivo and/or in vitro. An iterative approach consists in reviewing the gene regulatory networks at each cycle with novel biological predictions and new information brought by experimental methods. The cornerstone of the modelling process is the selection of parameter values that are consistent with the known properties of the system. In this article, the coherent parameter selection step of the iterative approach is illustrated in an extension of Thomas’ discrete modelling framework. This extension encodes into multiplexes information about cooperative, concurrent or more complex molecular interactions. We emphasize how formal methods can be helpful to perform the selection step. We express the dynamic knowledge into a temporal logic formula and we confront it, via model checking algorithm, to each model consistent with the static knowledge. In this way, all the models consistent with both static and dynamic knowledge are selected. The software platform SMBioNet implements this approach. We illustrate its use with a biological system triggering the tail resorption during the metamorphosis of tadpole.
Ahmed Mansour (Egypt), Jaime A. Teixeira da Silva (Japan), Gábor Gyulai (Hungary) Assessment of Molecular (Dis)similarity: The Role of Multiple Sequence Alignment (MSA) Programs in Biological Research (pp 23-30)
Mini-Review: Multiple sequence alignments (MSAs) are powerful tools in modern molecular biology that rely on sequence comparison methods. Based on MSAs, structural models, functional predictions, and phylogenetic trees can be created. MSAs are also used to infer the function of newly sequenced genes, predict new members of gene families, explore evolutionary relationships, sequence annotation, structural and functional predictions for genes and proteins. In this mini-review we emphasize the practical application of different MSA methods.
Tsuyoshi Hachiya, Yasubumi Sakakibara (Japan) Sensitive Detection of Conserved Gene Clusters Unravels the Evolutionary Forces behind the Correlation between Protein Sequence Homology and Gene Order Conservation (pp 31-45)
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Original Research Paper: A conserved gene cluster (also referred to as a conserved gene order) is defined as a cluster of neighboring genes whose gene order is conserved across several species. In the present study, we propose a novel workflow which enables sensitive detection of conserved gene clusters by taking into account the information of gene order conservation in the step to identify orthologous genes (OGs). Our workflow was applied to large-scale comparisons of 101 prokaryotic and 15 fungal genomes. Thereafter, we examined the difference between OGs in conserved gene clusters (clustered OGs) and OGs that are not the members of conserved gene clusters (isolated OGs). Our analysis confirms the finding in previous studies that, in prokaryotes, protein sequences of clustered OGs are more conserved than those of isolated OGs. In addition, this interesting correlation between protein sequence homology and gene order conservation were observed also in fungal genomes. To our knowledge, this is the first report of a systematic survey of such correlation in eukaryotic genomes. Furthermore, we analyzed evolutionary forces behind the correlation by estimating the rate of synonymous substitutions (KS) and the rate of nonsynonymous substitutions (KA). This detailed sequence analysis reveals that although the correlation is consistently observed and seems to be a general trend among prokaryotic and fungal genomes, the evolutionary forces behind the correlation are different among lineages, suggesting that the joint effect of heterogeneous underlying mechanisms would result in the correlation.
Ahmed Mansour (Egypt) Phylip and Phylogenetics (pp 46-49)
Research Note: Phylogenetics studies are mainly concerned with evolutionary relatedness among various groups of organisms. Recently, phylogenetic analyses have been performed on a genomic scale to address issues ranging from the prediction of gene and protein function to organismal relationships. Computing the relatedness of organisms either by phylogenetic (gene by gene analyses) or phylogenomic (the whole genome comparison) methods reveals high-quality results for demonstrating phylogenies. In this regard, Phylip (Phylogeny Inference Package) software is a free package of programs for inferring phylogenies of living species and organisms. It is now one of the most widely used packages for computing accurate phylogenetic trees and carrying out certain related tasks. This paper provides an overview on Phylip package and its applications and contribution to phylogenetic analyses.
Arumugam Chandrasekar, Aikkal Riju, Nambissan V. Sathyanath, Santhosh J. Eapen (India) SpicEST – An Annotated Database on Expressed Sequence Tags of Spices (pp 50-53)
Research Note: SpicEST is an attempt to develop a comprehensive database on ESTs of two spice plants, ginger and turmeric. For this, EST records were downloaded from NCBI open source database. All EST records for Curcuma longa and Zingiber officinale were mined and stored in MYSQL database. These ESTs were assembled using CAP3 sequence assembly program. The resultant contigs were stored in the database tissue- and crop-wise. By navigating the menus one can retrieve the results of SSRs identified from these ESTs by using five different SSR identification tools – MISA, ETRA, SSR PRIMER, SSRIT and WEB TROLL. Using these, SSR primers were designed with the help of PRIMER3 software and stored in the database after checking their quality using FAST PCR. Primers are being validated in wet lab studies. All the ESTs were annotated using the ESTPASS server. These results were used to find the putative genes and the respective metabolic pathways. The database also contains information on single nucleotide polymorphism in both these spices. Two different programs like CAP3 and AUTOSNP were used for this analysis and results can be retrieved by clicking on the respective tab. 'SpicEST' database is an initiative to promote spice genomics studies and is envisaged as an online tool for spice researchers. It is available as an online database at www.spices.res.in/spiceest.
Ming Li Wang, Noelle A. Barkley, Tracie M. Jenkins (USA) Microsatellite Markers in Plants and Insects. Part I: Applications of Biotechnology (pp 54-67)
Invited Review: Biotechnology is integral to the application of robust, high through-put detection of species-specific and species or genus-transferred microsatellites, or simple sequence repeat (SSR) markers. These short, tandemly repeated stretches of DNA of variable motifs and lengths are relatively evenly distributed throughout eukaryotic nuclear, chloroplasts, and mitochondrial genomes. Microsatellites are inherited as Mendelian co-dominant markers that provide insights into non-Mendelian inheritance such as microsatellite evolution, replication, repair, recombination, and mutation. These characteristics have made microsatellites the genetic marker of choice for most technologically-driven applications in plant and insect genetic studies such as mapping, marker-assisted selection (MAS), and genetic diversity studies. MAS and linkage mapping analyses has greatly assisted breeding programs through the discovery and isolation of many important agronomic genes that underlie respective phenotypes. Linkage maps and genome sequences have provided comparative genomic insights in plants and insects regarding microsatellite distribution, occurrence, and adaptive phenotypic evolution. Furthermore, genomic synteny and SSR sequence conservation have not only provided maximum annotated information for model plants and insects, but have demonstrated cross-species/genera transferability, which is indicative of long evolutionary history. It is the aim of this paper, therefore, to review biotechnology platforms and applications that have made SSR markers so useful as well as to discuss the impact of SSR transferability across species and/or genera.
Wenling Zhou, Rufang Tan, Chuanjun Xu, Yanyan Lai, Dongyin Chen, Ling Li (China) Gibberellic Acid Inhibits Browning, Enzyme Activity and Gene Expression of Phenylalanine Ammonia-Lyase in Phalaenopsis Leaf Explants (pp 68-71)
Original Research Paper: The effects of gibberellic acid (GA3) on browning, enzyme activity and gene expression of phenylanine ammonia lyase (PAL, EC 22.214.171.124) in Phalaenopsis (DoritaenopsisQueen Bee “Red Sky”) leaf explants during tissue culture were investigated. GA3 at 0 (control), 1, 5, 10, 25, 30, 50 and 70 mg L-1 were used to treat the explants. The percentage browning and tannin content of explants treated with 25 mg L-1 GA3 were far lower than of control explants cultured for more than 4 days. Paclobutrazol (PBZ), a gibberellin biosynthetic inhibitor, at 5 mg L-1, increased the browning percentage, which implied that it was endogenous GA3 that repressed browning. PAL activity and PAL gene expression were reduced by GA3 treatment but were increased by treatment with PBZ. These results indicate that GA3 is responsible for the browning of leaf explants through the regulation of PAL gene expression and enzyme activity in Phalaenopsis tissue culture.
Nariman A. H. Aly, Hussein A. Mona (Egypt) Novel Characteristics of Local Xenorhabdus and Photorhabdus Isolates with Phenotypic Heterogeneity, 16S rRNA Sequence Variation and High Toxicity to Galleria mellonella (pp 72-79)
Original Research Paper: Four local symbiotic bacterial isolates comprised of three Photorhabdus luminescens andone Xenorhabdus nematophila isolated from nematodes (Heterorhabditis bacteriophora and Steinernema carpocapsae, respectively) that were previously characterized by their high toxicity to Galleria mellonella, were characterized. The nematodes were isolated from the soil samples collected from four Egyptian governorates using Galleria larvae as bait. Cultural properties as well as cellular morphology of primary and secondary form variants are discussed. Four forward primers within the variable domain of the 16S rRNA gene at position 440 to 480 bp and the reverse primer from the highly conserved region at 755 to 795 bp were used to amplify a 355-bp fragment. Primer TRO displayed the fragment in the three P. luminescens isolates, while it was absent in the X. nematophila isolate. On the contrary, primer TEM showed the fragment in the X. nematophila isolate and the gene was undetected in the three P. luminescens isolates. The two other primers revealed the fragment in all isolates. Sequence alignment revealed 32 nucleotide positional differences between the novel local Photorhabdus isolate (FJ755891) and the numerous isolates and strains of P. luminescens and X. nematophila based on the 16S rRNA gene similarity. Phylogenetic analysis based on 16S rRNA gene sequences showed that the local isolate (FJ755891) formed a phylogenetically distinct group, separate from all other named isolates and species of P. luminescens and X. nematophila. Such obtained results evidently indicated a large diversity with unique characteristics of the local Egyptian isolates from all the other isolates and strains established around the world. Two of the four specific primers detected the 16S rRNA gene with 355 bp in the four isolates, while the two other primers displayed the gene in either one and three isolates belonging to P. luminescens.
Heba Amin, Osama E. El-Sayed, Khaled El-Dougdoug, Badawy Othman, Mostafa A. M. Gomaa (Egypt) PCR-Detection of Ry and Rx Genes from Potato with Potential for Wide Applications in Plants (pp 80-88)
Original Research Paper: Nine potato cultivars (‘Lady Rosetta’, ‘Spunta’, ‘Burren’, ‘Cara’, ‘Hermine’, ‘Nicola’, ‘Hermes’, ‘Draga’ and ‘Diamond’) and five wild species (Solanum tuberosum andigena, S. acaule, S. demissum, S. hougasii and S. stoloniferum) were cultivated over three successive seasons from 2004 to 2008 and were systemically infected with potato virus y (PVY) and potato virus X (PVX). DAS-ELISA detected the presence of both viruses in 9 inoculated cultivars and 5 wild species and the infected plants were categorized according to their degree of infection. Among the five designed primers, the 242 bp primer detected the Ryadg gene in five cultivars and S. stoloniferum and five primers detected the Ryadg genein S. andigena. One of the two designed primers with 541 bp detected the Ryfsto gene in six cultivars. The Rx gene was detected in four cultivars, S. acaule and S. andigena using six designed primers. The existence of Ryadg and Rx genes among ‘Lady Rosetta’, ‘Cara’, ‘Diamond’, ‘Nicola’, ‘Draga’ and ‘Spunta’ and the wild species were confirmed using 242 and 241 bp fragments as probes, respectively by dot-blot hybridization, which showed similar results. Two fragments of PCR products with 201 and 456 bp corresponding to Rx and Ry genes, respectively were sequenced, analyzed and submitted to GenBank under EU687573 and EU687574.
Mohamed Najeb Barakat, Adel Ahamed El-Shafei, Abdullah Abdulaziz Al-Doss (Saudi Arabia) Identification of Molecular Markers Linked to Northern Corn Leaf Blight Resistance in Yellow Population of Maize (pp 89-95)
Original Research Paper: The objectives of this study were to identify RAPD, ISSR and SCAR markers linked to northern corn leaf blight (NCLB) resistance genes in an F2 population of yellow maize (Zea mays L.) using bulked segregant analysis and to map NCLB resistance genes in F2 populations of maize. The F2 yellow population of maize was developed from a cross between the resistant line Gm1021 and the susceptable line Gm1002. Bulked segregant analysis with RAPD, ISSR and SCAR markers was conducted to identify markers that were linked to the Helminthosporium turcicum (Ht) gene. The genetic distance between RAPD markers (OPC04220 bp, OPC04450 bp and OPE20270 bp) and NCLB Ht resistance genes were 2.1, 7.8 and 13.4 cM, respectively, with logarithm of odds (LOD) scores of 34.2, 23.1, and 23.5, respectively. Therefore, these three RAPD markers were linked to the quantitative trait loci (QTLs) for the NCLB Ht resistance gene. The genetic distance between the SCAR marker (SCE20270 bp), which was derived from the converted OPE20270 bp RAPD marker, and the NCLB Ht resistance gene, was 2.1 cM with an LOD score of 34.2. This confirmed that the SCAR marker targeted the same locus as the RAPD marker and that it too was linked to the QTL for the NCLB Ht resistance gene. The genetic distance between an ISSR marker (HB13200 bp) and the NCLB Ht resistance gene was 1.4 cM with an LOD score of 32.5. Therefore, this ISSR marker was linked to the QTL for the NCLB Ht resistance gene. The present study indicates that RAPD, ISSR and SCAR markers, combined with bulked segregant analysis, can be used to identify molecular markers linked to the NCLB resistance gene in maize. Once these markers are identified, they can be used to detect the QTLs linked to NCLB resistance in breeding programs, as a selection tool in early generations.
Satender Rana, Poonam Shirkot, M. C. Yadav (India) A Female Sex Associated Randomly Amplified Polymorphic DNA Marker in Dioecious Hippophae salicifolia (pp 96-101)
Original Research Paper: In many dioecious plants, gender influences economic value, breeding schemes and opportunities for commercial harvest. In the plant kingdom, dioecism has arisen independently in different families and genera, and several distinct genetic mechanisms regulating dioecy have been found. Molecular studies are beginning to detail the genetic control of dioecy in several plant species. However, there is paucity of information on chromosomal and genetic basis of sex of gender determination. RAPD markers were used to differentiate between pistillate and staminate genotypes of Hippophae salicifolia. Out of the 31 decamer primers used, 27 primers amplified the genomic DNA of all the 10 genotypes taken for studies. 25 primers were found to be polymorphic. In total, 118 DNA bands were reproducibly obtained, out of which 95 (80.5%) were polymorphic. The polymorphism were scored and used in band-sharing analysis to identify genetic relationships. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA grouped all the 10 genotypes into two major groups. Similarity indices ranged from 0.26 to 0.82. A female-specific RAPD marker of mol. wt. 1190 bp was obtained with primer OPF-11. This RAPD marker specific for female genotype can be utilized in the future to develop a SCAR (sequence characterized amplified region) marker. This is the first report on gender identification in Hippophae salicifolia using RAPD markers.
Hesham M. Abdullah, Fawzy A. El-Feki (Egypt) Molecular Evolution of Hemagglutinin (HA) Gene of H5N1 Avian Flu Virus Isolated from Chickens, Ducks and Human Cases in Egypt (pp 102-109)
Original Research Paper: The molecular phylogenetic tree of the Hemagglutinin (HA) gene within and between (H5N1) viral isolates from birds and human cases in Egypt was constructed and analyzed by using Laser gene 7.1 software. The HA gene data set was downloaded from GenBank in January 2008. Results showed that teal duck H5N1 virus isolates (GenBank accession nos. EF042623 and EF042624) formed the root of the phylogenetic tree of all H5N1 isolates within and between hosts. The human H5N1 isolate (GenBank accession nos. EF535825), which was isolated from El-Menia governorate, was the root of all infected human cases while chicken isolates, which originated in Cairo and Giza governorates, in addition to the WHO strain (GenBank accession nos. DQ837588, DQ837589 and EU146879, respectively) were found to be the roots of the phylogenetic tree of infected chickens. The HA gene had varied sequence length (bp) in the different viral isolates ranging between 1704 and 1743 bp. These results suggest that the outbreak of avian flu infection in Egypt, which had first been reported in 2005, was transmitted by migrating birds, especially teal ducks, and that infection was concentrated only in the east side of Egypt extending from Damietta to Aswan rather than the west side of Egypt based on the survey of HA gene flat files which were submitted to GenBank. The H5 HA genes have high mutation and reassortment rates which evolved rapidly due to their circulation in birds and humans. The authors suggest that the H5N1 isolates of Cairo, Giza and El-Menia (GenBank accession nos. DQ837588, DQ837589, EU183325 and EF535825, respectively) will be good candidates for vaccine production.
Subramanian Ganesh Manikandan, Cuntheepuram Lakshminarasimhan, Nooruddin Thajuddin, Ramalingam Saravanan, Gangatharan Muralitharan (India) Amplification of Methicillin Resistance Gene (mec-A) from Staphylococcus aureus Isolates from India (pp 110-112)
Research Note: Staphylococcus aureus strains were isolated from 261 clinical samples located at various regions of Tamil Nadu, India. All isolates were grown properly using standard methodology and were used for the isolation of chromosomal DNA. The chromosomal DNA was used for the confirmation of a methicillin resistant gene named mec-A. The strains possessing the mec-A gene were highly resistant to the antibiotic, methicillin. The mec-A gene (size = 310 bp) was successfully detected by Real Time-PCR.