Set Of Degenerate Primers Research Articles

Isolation and characterization of NBS–LRR resistance gene analogues from mango

The nucleotide-binding site (NBS)–leucine-rich repeat (LRR) gene family is a class of R genes in plants. NBS genes play a very important role in disease defence. To further study the variation and homology of mango NBS–LRR genes, 16 resistance gene analogues (RGAs) (GenBank accession number HM446507-22) were isolated from the polymerase chain reaction fragments and sequenced by using two degenerate primer sets. The total nucleotide diversity index Pi was 0.362, and 236 variation sites were found among 16 RGAs. The degree of homology between the RGAs varied from 44.4% to 98.5%. Sixteen RGAs could be translated into amino sequences. The high level of this homology in the protein sequences of the P-loop and kinase-2 of the NBS domain between the RGAs isolated in this study and previously characterized R genes indicated that these cloned sequences belonged to the NBS–LRR gene family. Moreover, these 16 RGAs could be classified into the non-TIR–NBS–LRR gene family because only tryptophan (W) could be claimed as the final residual of the kinase-2 domain of all RGAs isolated here. From our results, we concluded that our mango NBS–LRR genes possessed a high level of variation from the mango genome, which may allow mango to recognize many different pathogenic virulence factors.

Analysis of the scallop microbiota by means of 16S rRNA gene pyrosequencing

Event Abstract Back to Event Analysis of the scallop microbiota by means of 16S rRNA gene pyrosequencing Aide Lasa1, Alex Mira2, A Camelo-Castillo2, P. Belda-Ferre2 and Jesus L. Romalde1* 1 Universidad de Santiago de Compostela, Spain 2 Centro Superior de Investigación de la Salud Pública (CSISP-FISABIO, Departamento de Genómica y Salud,, Spain Bivalve molluscs, due to its filter-feeding mechanism, have an abundant associated bacterial microbiota composed of a large number of species of different genera. The knowledge about the composition of the microbiota associated with bivalve molluscs is based, mostly, on techniques of cultivation of microorganisms, but also on DGGE methods. However, it is estimated that less than 1% of the bacteria are culturable with current methods, which lead to an underestimation of the microbial diversity. On the other hand, DGGE methodology has a limited resolution due to the low number of DNA fragments obtained as representative of a microbial community. The introduction of new molecular approaches allowed the improvement of the knowledge about bacterial communities present in a sample [1]. In recent years, new molecular techniques known as Next Generation Sequencing (NGS) have been developed, based on high- throughput sequencing [2]. In this study, we present the analysis of the microbiota associated to reared scallop gonads before and after spawning by pyrosequencing of the 16S rRNA gene. After the DNA extraction of the samples, amplification was performed using a set of degenerate primers with low stringency conditions. Pyrosequencing of samples was carried out using the sequencer 454 GLX Titanium Plus (Roche). The data obtained was further subjected to bioinformatics analysis using Galaxy and UCHIME program and the Ribosomal Database Project. Rarefaction curves were obtained, the composition of the bacterial community was analysed after the taxonomic assignment, and the total composition of the bacterial communities of the samples were compared using the principal coordinates analysis (PCoA) by FastUnifrac tool [3]. Pyrosequencing of the samples resulted in a total of 18520 sequences (3000 per sample, approximately) with an average length of 325 bp (base pairs). The taxonomic assignment of sequences allowed the identification to the genus level, being observed a large bacterial diversity with over 110 genera. The most prevalent genera in the samples were Hydrotalea, Acinetobacter, Delftia, Sediminibacter and Pseudomonas, among others. Differences in the microbial communities were observed among the samples, and the PCoA analysis allowed their separation by means on their gender and if they proceed from sampling before or after the spawning. Nevertheless, the rarefaction curves obtained for each sample failed to reach a saturation phase, indicating that more sequencing effort would be necessary.

RETRACTION of: Down-regulation of protein tyrosine phosphatase gene expression in lactating mouse mammary gland

Detailed analysis of protein tyrosine phosphatase (PTP) expression in mouse mammary gland and mammary epithelial cells using a set of degenerate primers corresponding to the PTP core domain sequence revealed the presence of 16 different receptor-type and intracellular PTPs. Northern blot and RT-PCR analyses revealed that some PTPs were up-regulated during gestation, suggesting that these enzymes are involved in development of mammary gland. However, expression of most PTPs dramatically decreased during lactation, whereas the beta-casein gene expression was increased and remained at a high level. At the involution stage after weaning, most PTPs were up-regulated and their expression returned almost to the virgin level. Such up-regulation was also induced by forced weaning in lactating mother mice. These results suggest the possible contribution of PTPs to the development, involution, and remodeling of mammary gland and their possible inhibitory action on maintaining high expression of milk genes during lactation.

Utilization of Heme as an Iron Source by Marine Alphaproteobacteria in the Roseobacter Clade

The bioavailability and utilization of porphyrin-bound iron, specifically heme, by marine microorganisms have rarely been examined. This study used Ruegeria sp. strain TrichCH4B as a model organism to study heme acquisition by a member of the Roseobacter clade. Analogs of known heme transporter proteins were found within the Ruegeria sp. TrichCH4B genome. The identified heme uptake and utilization system appears to be functional, as the heme genes were upregulated under iron stress, the bacterium could grow on ferric-porphyrin complexes as the sole iron source, and internalization of (55) Fe from ferric protoporphyrin IX was observed. The potential ability to utilize heme in the Roseobacter clade appears to be common, as half of the isolates in the RoseoBase database were found to have a complete heme uptake system. A degenerate primer set was designed and successfully used to identify the putative heme oxygenase gene (hmus) in the roseobacter heme uptake system from diverse nonenriched marine environments. This study found that members of the Roseobacter clade are capable of utilizing heme as an iron source and that this capability may be present in all types of marine environments. The results of this study add a new perspective to the current picture of iron cycling in marine systems, whereby relatively refractory intracellular pools of heme-bound iron may be taken up quickly and directly reincorporated into living bacteria without previous degradation or the necessity of a siderophore intermediate.

Sequence diversity and expression levels of Synechococcus phosphate transporter gene in the East China Sea

The phosphate-binding transporter gene (pstS) is a potential diagnostic marker for inorganic phosphorus stress in picocyanobacteria. In order to evaluate pstS messenger (m)RNA levels in the East China Sea (ECS) by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), suitable primer sets had to be designed according to local gene diversity. For this purpose, 71 homologous pstS sequences were collected during 3 cruises conducted in March 2006, and April and July 2007. The phylogenetic analysis indicated that all pstS sequences obtained from the continental shelf region in the northern ECS belonged to Synechococcus clades II, III, and V. In contrast, only a small fraction of pstS sequences obtained in the Kuroshio region was from Synechococcus. According to sequence alignment, a degenerate primer set was designed for Synechococcus. The specificity of the primers was confirmed using genomic DNA extracted from several Synechococcus and Prochlorococcus cultured strains. Subsequently, in the spring seasons of 2007 and 2008, the pstS mRNA abundance was measured by the Synechococcus-specific primer set at a station in the oligotrophic Kuroshio Current adjacent to the ECS. It was found that the pstS expression pattern was inversely correlated with the concentration of inorganic phosphate in surface waters, which closely resembled an analogous relationship in a cultured Synechococcus. A similar relationship was formed between pstS expression and the previous-day aerosol particles, but nonexistent regarding mixed layer depths.

Detection of Avian coronavirus infectious bronchitis virus type QX infection in Switzerland

Infectious bronchitis, a disease of chickens caused by Avian coronavirus infectious bronchitis virus (IBV), leads to severe economic losses for the poultry industry worldwide. Various attempts to control the virus based on vaccination strategies are performed. However, due to the emergence of novel genotypes, an effective control of the virus is hindered. In 1996, a novel viral genotype named IBV-QX was reported for the first time in Qingdao, Shandong province, China. The first appearance of an IBV-QX isolate in Europe was reported between 2003 and 2004 in The Netherlands. Subsequently, infections with this genotype were found in several other European countries such as France, Italy, Germany, United Kingdom, Slovenia, and Sweden. The present report describes the use of a new set of degenerate primers that amplify a 636-bp fragment within the S1 gene by reverse transcription polymerase chain reaction to detect the occurrence of IBV-QX infection in Switzerland.

Assessment and modification of degenerate qPCR primers that amplify functional genes from etheneotrophs and vinyl chloride-assimilators

Degenerate qPCR primer sets that target the functional genes etnC and etnE in etheneotrophs and vinyl chloride-assimilating bacteria were assessed and modified in an effort to improve performance. Functional gene abundance in four pure cultures was estimated by qPCR using novel (MRTC and MRTE) and existing (RTC and RTE) degenerate primer sets and compared to abundances estimated with nondegenerate gene-specific primers (GSPs). Functional gene abundance in groundwater DNA extracted from several contaminated sites was also estimated with MRTC and MRTE primers. MRTC primers displayed significantly improved etnC quantification in both pure cultures and environmental samples. Application of MRTC and MRTE primer sets will enhance microbial ecology studies involving etheneotrophs and qPCR analyses that support vinyl chloride bioremediation strategies.

Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers

Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89–150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ≥95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays.

Cloning, expression and functional characterization of carp,Cyprinus carpio, estrogen receptors and their differential activations by estrogens

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in vertebrates and promote the growth and differentiation of the female reproductive system. Importantly, environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor ligand interactions in the carp, Cyprinus carpio, a species used widely for both field- and laboratory-based studies, we cloned all three carp estrogen receptors (ER; ERα, ERβ1 and ERβ2) and applied an estrogen-responsive (ERE)-luciferase reporter assay system to characterize the interactions of these receptors with steroidal and synthetic estrogens. DNA fragments encoding all three ERs in carp, ERα, ERβ1 and ERβ2, were obtained from the ovary using degenerate primer sets and PCR techniques, and full-length carp ER (cER) cDNAs were then obtained using RACE (rapid amplification of the cDNA end) techniques. Amino acid sequences of cERs showed overall homology of 46% (α vs β1), 49% (α vs β2) and 53% (β1 vs β2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen-dependent activation of transcription and cERβ2 showed a higher sensitivity to the natural steroid oestrogen, 17β-estradiol, than cERα. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptor-environmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution.

High Genetic Diversity and Different Distributions of Glycosyl Hydrolase Family 10 and 11 Xylanases in the Goat Rumen

BackgroundThe rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment.Methodology/Principal FindingsWe explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11) in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95%) were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family) were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific.Conclusion/SignificanceThe numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen microenvironment.

Degenerate primers for PCR amplification and sequencing of the avian influenza A neuraminidase gene

This study describes the design of degenerate primers and their use for synthesis of full-length avian influenza A neuramindase (NA). Each reaction was performed using either two forward primers and one reverse primer, or one forward primer and one reverse primer. Both primer combinations had comparable amplification efficiencies for all NA subtypes (1–9). A total of 115 virus strains, including both field isolates and reference strains, were amplified successfully using these degenerate primer sets. Of the sequences amplified, 108 strains (93.9%) resulted in near full-length NA cDNAs after two readings with one forward primer and one reverse primer. Of the remaining sequences, five strains (4.3%) yielded reads with enough information for subtype categorization by BLAST although they were of insufficient quality for assembly. One strain (0.9%) yielded different subtypes from both sequence reads whereas the other one (0.9%) was not possible to assemble and subtype. This successful demonstration of these degenerate primers for the amplification and sequencing of all avian NA subtypes suggests that these primers could be employed in the avian influenza surveillance program as well as studies of antiviral resistance, virus ecology or viral phylogeny.

Molecular detection and diversity of xylanase genes in alpine tundra soil

Xylan is a major polysaccharide in plant cell walls, and its degradation is mainly conducted by microbial xylanases in nature. To explore the xylanase diversity in the environment, two sets of degenerate primers were designed based on the microbial xylanase sequences in Pfam database of glycosyl hydrolase (GH) family 10 and 11 and were used to amplify objective gene fragments directly from the alpine tundra soil DNA of the Tianshan Mountains, China. Ninety-six distinct GH 10 and 31 GH 11 xylanase gene fragments were retrieved, and most of them have low identities with known sequences in GenBank. Based on phylogenetic analysis, all of the GH 10 xylanase sequences fell into six clusters and were related to xylanases from Actinobacteria, Proteobacteria, Verrucomicrobia, Bacteroidetes, Firmicutes, and Acidobacteria. Three clusters of GH 11 xylanase sequences were established, and two of them were related with enzymes from fungi. These results indicated the diversity of xylanase genes in this cold environment. Four xylanolytic strains were isolated from the soil, and GH 10 xylanase gene fragments were cloned using the same primers. A full-length gene was obtained and expressed in Escherichia coli, and the recombinant enzyme showed some cold-related characteristics. Our study provides an efficient molecular approach to study xylanase in complex environments and casts an insight into the diversity and distribution of xylanases in a cold environment, which is very meaningful to understand their roles in xylan degradation in nature.

First Report of Bean yellow mosaic virus in Alaska from Clover (Trifolium spp.)

In mid-June 2008, distinct mosaic leaves were observed on a cluster of clover (Trifolium spp.) with light pink and white flowers growing at the edge of a lawn in Palmer, AK. Virus minipurification from leaves of affected clover and protein extractions on a polyacrylamide electrophoresis implicated a ~35-kDa putative coat protein (CP). Subsequent western blots and ELISA with a universal potyvirus antiserum (Agdia Inc., Elkhart, IN) confirmed potyvirus identity. Total RNA extracts (RNeasy Plant Mini Kit, Qiagen Inc., Valencia, CA) from the same plant were used for reverse transcription (RT)-PCR. Three sets of degenerate primers that targeted potyvirus-specific genes, HC-Pro (helper component protease) and CI (cylindrical inclusion protein) and the genomic 3'-terminus that included a partial NIb (nuclear inclusion), CP (coat protein), and UTR (untranslated region), produced the expected PCR segments (~0.7, ~0.7, and ~1.6 kbp, respectively) on 1% agarose gels (1). Direct sequencing of the HC-Pro (GenBank No. GQ181115), CI (GQ181116), and CP (GU126690) segments revealed 98, 97, and 99% nucleotide identities (no gaps), respectively, to Bean yellow mosaic virus (BYMV)-chlorotic spot (CS) strain, GenBank No. AB373203. The next closest BYMV percent identity comparisons decreased to 79% for HC-Pro (GenBank No. DQ641248; BYMV-W), 79% for CI (U47033; BYMV-S) partial genes, and 96% for CP (AB041971; BYMV-P242). Mechanical inoculations of purified virus preparations produced local lesions on Chenopodium amaranticolor Coste & A. Reyn. (2 of 5) and C. quinoa Willd. (6 of 7), and mosaic on Nicotiana benthamiana Domin (5 of 5). BYMV was specifically confirmed on tester plants using a double-antibody sandwich (DAS)-ELISA BYMV (strain 204 and B25) kit (AC Diagnostics, Inc., Fayetteville, AR) as directed. The absence of another potyvirus commonly found in clover, Clover yellow vein virus (ClYVV), was verified in parallel DAS-ELISA ClYVV assays (AC Diagnostics, Inc). The BYMV isolate was maintained in N. benthamiana, and virion or sap extracts inoculated to the following host range (number of infected/total inoculated plants [verified by BYMV ELISA]): Cucumis sativus L. 'Straight Eight' (0/5), Gomphrena globosa L. (1/4), Nicotiana clevelandii A. Gray (4/7), Phaseolus vulgaris L. 'Bountiful' (1/3), Pisum sativum L. (Germplasm Resources Information Network Accession Nos. -PI 508092 (8/12), -W6 17525 (13/13), -W6 17529 (0/13), -W6 17530 (13/14), -W6 17537 (0/12), -W6 17538 (0/12), and -W6 17539 (0/21), Tetragonia tetragoniodes (2/2), Trifolium pretense L. 'Altaswede' (6/10), T. repens L. 'Pilgrim' (0/8), and Vicia faba L. (1/3). All infected plants had symptoms ranging from systemic mosaic (T. pretense, P. sativum) to leaf distortions (N. clevelandii, Tetragonia tetragoniodes). Interestingly, the host range and genomic sequences of the BYMV Alaskan strain resemble the BYMV-CS (chlorotic spot) strain that was originally isolated from a diseased red clover (T. pretense) plant in Japan more than 40 years ago (2). Although BYMV occurs worldwide and has a wide host range in dictoyledonous and monocotyledonous plants (3), to our knowledge, this is the first report of a natural occurrence of BYMV in Alaska. The incidence and distribution of BYMV in clover and other plant species are not known in Alaska.

Amplification of polyketide synthase gene fragments in ochratoxigenic and nonochratoxigenic black aspergilli in grapevine

Members of the Aspergillus section Nigri , also known as black aspergilli produce Ochratoxin A (OTA), a contaminant of wine. Despite potentially severe health effects and economic losses caused by OTA in wine, almost nothing is known about the genetics that lies behind its biosynthesis in black aspergilli in grapevine. In this work, degenerate primer sets were used to amplify 49 Polyketide Synthase (PKS) gene fragments in reference strains of A. carbonarius , A. niger and A. tubingensis . Deduced amino acid sequences were then compared with those of aolc35-12 and aoks1 , two PKS genes involved in OTA biosynthesis in A . westerdijkiae. A putative homologue of aolc35-12 was found in A. carbonarius (63% amino acid identity), the main OTA producer on grapes and in an ochratoxigenic A. niger strain (58%). In A. niger this fragment corresponded to an15g07920, a PKS already annotated in the sequenced A. niger CBS 513.88 genome as putatively involved in OTA biosynthesis. No aolc35-12 candidates were found in atoxigenic A. tubingensis isolates and no putative homologues of aoks1 were found in any of the screened strains. A screening of A. niger field isolates using specific primers for an15g07920 indicated that the absence of this gene is apparently related to a failure to produce OTA. The present work gives a first insight into the genetics of OTA biosynthesis in black aspergilli in grapevine and represents a starting point for further investigation of the OTA biosynthesis pathway and the development of molecular methods to detect the producers in vineyards.

Polymerase Chain Reaction-based Detection of Total and Specific Vibrio Species

The polymerase chain reaction (PCR) technique is widely used for efficient detection of food-borne pathogens because of speed and specificity. However, PCR methods have focused mostly on species-specific detection. In the present work, we describe a PCR-based method for the simultaneous detection of all Vibrio species because lots of them are notorious food-borne human pathogens. We then combined this total detection method with specific detection of Vibrio cholerae pathogen. Using a degenerate primer set based on the sequence of the potassium uptake gene, trkA, we were able to successfully detect all Vibrio species. Specific detection of V. cholerae was also possible using primer sets based on putative flagellin sequence. Importantly, simultaneous total and species-specific Vibrio detection was possible using all two primer sets in a multiplexed PCR strategy. Thus, the PCR method we have developed is applicable to both simultaneous and two-step detection of total and specific Vibrio species.

Cloning of a New Xylanase Gene from Streptomyces sp. TN119 Using a Modified Thermal Asymmetric Interlaced-PCR Specific for GC-Rich Genes and Biochemical Characterization

A bacterial strain, Streptomyces sp. TN119, was isolated from the gut of Batocera horsfieldi larvae and showed xylanolytic activity. A degenerate primer set was designed based on the base usage of G and C in Actinobacteria xylanase-coding sequences belonging to the glycosyl hydrolases family 10 (GH 10), and used to clone the partial xylanase gene from Streptomyces sp. TN119. A modified thermal asymmetric interlaced (TAIL)-PCR specific for high-GC genes, named GC TAIL-PCR, was developed to obtain the full-length xylanase gene (xynA119; 1089 bp). Rich in GC content (67.8%), xynA119 encodes a new GH 10 xylanase (XynA119), which shares highest identity (48.8%) with an endo-1,4-beta-xylanase from Cellulosimicrobium sp. HY-12. Recombinant XynA119 was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 6.5 and 60 degrees Celsius, was stable at pH 4.0 to 10.0 and 50 degrees Celsius, was resistant to most chemicals (except for Cu(2+), Mn(2+), Ag(+), Hg(2+) and SDS) and trypsin, and produced simple products. The specific activity, K(m), V(max), and k(cat) using oat-spelt xylan as substrate were 57.9 U mg(-1), 1.0 mg ml(-1), 74.8 micromol min(-1) mg(-1), and 49.2 s(-1), respectively.

A New Aminopeptidase from the Keratin-Degrading Strain Streptomyces fradiae var. k11

An aminopeptidase gene fragment was isolated from a keratin-degrading strain, Streptomyces fradiae var. k11, by PCR amplification using a degenerate primer set designed based on the partial amino acid sequence of the native enzyme. The gene, designated sfap, encoded a polypeptide of 461 amino acids comprised of three domains: a signal peptide, a mature region, and a C-terminal propeptide. The aminopeptidase, SFAP, had highest amino acid sequence identity (79%) with a putative aminopeptidase from Streptomyces griseus subsp. griseus NBRC 13350. The gene with and without C-terminal propeptide was successfully overexpressed in Escherichia coli BL21 (DE3), and the gene without C-terminal propeptide encoded a functional enzyme. Purified recombinant SFAP exhibited optimal activity at pH 8.0 and 60 degrees C, and retained >60% peak activity over a broad range of temperature. The enzyme was thermal and pH stable, and showed metalloprotease characteristics, which was inhibited by EDTA but activated by Ca(2+) and Co(2+). This is the first study to report the gene cloning and expression of a leucine aminopeptidase from S. fradiae.

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing was developed by a synthesis method (DP-SBS) for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number (n) of degenerate nucleotides at the 3'-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides (n+1) on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.

Incidence of Viruses and Nematode Vectors in Lebanese Vineyards

AbstractSurveys for virus diseases and nematode vectors were conducted in 95 commercial vineyards of four different Lebanese districts (Bekaa valley, Mount Lebanon, North and South Lebanon). Out of 915 randomly collected grapevine samples tested by ELISA, 511 (55.8%) were infected by one or more viruses. Grapevine virus A (30.9%) and Grapevine leafroll‐associated virus 3 (23.7%) were the prevailing viruses, followed by Grapevine fleck virus (15.1%), Grapevine leafroll‐associated virus 1 (10.6%) and Grapevine leafroll‐associated virus 2 (8.7%). Arabis mosaic virus was not found whereas Grapevine fanleaf virus (GFLV) and Grapevine virus B were little represented. The most important Lebanese grapevine varieties, i.e. Maghdouchi, Tfeifihi and Beitamouni, had average infection rates between 70% and 87%, whereas varieties of foreign origin had a better sanitary status with the exception of cvs Cinsaut and Thompson (c. 83% infection). Grapevine rupestris stem pitting‐associated virus was detected in 79 of 90 (87.8%) samples tested by RT‐PCR and closteroviruses were recorded in seven of 70 (10%) vines tested. One of these viruses was identified as Grapevine leafroll‐associated virus 5 by ELISA and partial genome sequencing. No nepoviruses other than GFLV were detected in any of 90 samples tested using three different sets of degenerate primers. Xiphinema index was found in 23 of 89 soil samples collected from vineyards, and in three of 15 samples collected primarily under fig trees in fields where no grapevines were grown.

Rapid determination of the nucleotide sequences of potyviral coat protein genes using semi-nested RT-PCR with universal primers

A rapid method (<20 h) for determining the nucleotide sequences of potyviral coat protein (CP) genes was developed. The procedure involves reverse transcription using total RNA from a potyvirus-infected plant, followed by polymerase chain reaction (PCR) with the universal primers Sprimer/M4. The expected fragment, purified from an agarose gel, is used in semi-nested PCR with three degenerate primer sets: Sprimer/D1000, Sprimer/RYAFDFY-C and U341/M4. The three purified fragments are directly sequenced using the respective primers D1000, RYAFDFY-C and U341, then the complete CP sequence is assembled. All 11 potyviruses tested were efficiently sequenced by this method.