New Set Of Primers Research Articles

Molecular Biology Techniques Q & A

Molecular Biology Techniques Q & A

Molecular differentiation ofMycosphaerellaspecies fromMusa

The three Mycosphaerella species M. fijiensis, M. musicola and M. eumusae, that infect bananas, cause major problems to the exploitation of this crop around the world. Their differentiation is crucial to the understanding of their epidemiology and control. M. eumusae was only recognised as a species in 2000, before which the other two were known to occur on Musa. It is now very commonly present in several countries. Bananas being a major source of revenue for low-income countries, they remain an important export commodity for these developing countries. The genetic relatedness of these three species was investigated through sequence analysis of conserved genes, actin, tubulin and histone 3. All confirmed M. eumusae as being different from the other two. Previous data relied on ribosomal sequences. Primers developed by Johanson and Jeger (1993) and which are specific for M. musicola and M. fijiensis, were tested on all three species. M. eumusae had an amplification profile which allowed its differentiation from the other two. The amplified fragments from the three species were sequenced and sequence polymorphism was used for designing new primers. Two new sets of primers, EuY1/EuY5 and EuY2/EuY5, reported here produced profiles that consistently gave the same bands which were specific for the three species and can be used in conventional PCR for their differentiation.

PCR-based detection of bioluminescent microbial populations in Tyrrhenian Sea

Abstract The present study is focused on the development of a cultivation-independent molecular approach for specific detection of bioluminescent bacteria within microbial communities by direct amplification of luxA gene from environmental DNA. A new set of primers, specifically targeting free-living bioluminescent bacteria, was designed on the base of l uxA sequences available from the public database. Meso- and bathypelagic seawater samples were collected from two stations in Tyrrhenian Sea at the depths of 500 and 2750 m. The same seawater samples also were used to isolate bioluminescent bacteria that were further subjected to luxA and 16S rRNA gene sequencing. PCR products obtained by amplification with designed primers were cloned, and the phylogenetic affiliation of 40 clones was determined. All of them were clustered into three groups, only distantly related to the Photobacterium phosphoreum and Photobacterium kishitanii clades. The half of all clones formed a tight monophyletic clade, while the rest of clones were organized in “compartment”-specific, meso- and bathypelagic ecotypes. No matches with luxA gene sequences of four bioluminescent strains, isolated from the same seawater samples, were observed. These findings indicate that the PCR-based approach developed in present manuscript, allowed us to detect the novel, “yet to be cultivated” lineages of bioluminescent bacteria, which are likely specific for distinct warm bathypelagic realms of Mediterranean Sea.

Detection of Meat Species in Food Using a Single Primer Pair by PCR and a Novel DNA Extraction by Short Microwave Irradiation

A unique DNA isolation protocol based on microwave irradiation and density centrifugation for the simultaneous detection of porcine, chicken, and mutton meat species in food by PCR using newly developed primers is described. Emphasis is placed on the detection of pork, as it is considered unlawful in the diet in major religions such as Islam and Judaism. The method has been optimized using the primers designed from porcine mitochondrial DNA (mtDNA) sequence and tested in 96 samples. The method involves 35 seconds of high microwave irradiation of lysed sample homogenate followed by a 5- min centrifugation in 1.5 mL Eppendorf tubes until a clear supernatant is obtained. The design of this protocol makes it possible to process many samples in a short time. To evaluate the method pork and food spiked with different concentrations of pork together with unknown food samples were analyzed by PCR using the new set of porcine specific primers. The new set of primers and the method involved showed high sensitivity in detecting porcine and chicken content in food in addition as a marker for the detection of mutton. The present sample preparation method has the potential to be applied to other meat detection systems as well.

MAD-DPD: Designing Highly Degenerate Primers with Maximum Amplification Specificity

This work introduces minimum accumulative degeneracy, a variant of the degenerate primer design problem, which is particularly useful when a large number of sequences are to be covered by a set of restricted number of primers. A primer set, which is designed on a minimum accumulative degeneracy basis, especially helps to reduce nonspecific PCR amplification of undesired DNA fragments, as fewer primer species are present in PCR. A Boltzmann machine is designed to solve the minimum accumulative degeneracy degenerate primer design problem, called the MAD-DPD Boltzmann machine. This algorithm shows great flexibility, as it can be determined either to solve the problem with strict fidelity to covering all input sequences or to exclude some input sequences if it results in less degenerate primers. This Boltzmann machine is successfully implemented in designing a new set of primers for amplification of antibody variable fragments from mouse spleen cells, which theoretically covers more diverse antibody sequences than currently available primers. The MAD-DPD Boltzmann machine is available online at bioinf.cs.ipm.ir/download/MAD_DPD08172007.zip.

Duplex — reverse transcription — polymerase chain reaction (D-RT-PCR)-a technique for the simultaneous detection of viruses causing sugarcane mosaic

Sugarcane mosaic virus (SCMV) and Sugarcane streak mosaic virus (SCSMV) are the two major RNA viruses that are widely prevalent in most of the Asian countries including India and are the causative viruses of mosaic in sugarcane. We have established that these two viruses either alone or in combination cause mosaic in sugarcane through RT-PCR. Further studies were conducted to detect the associated viruses in a single reaction. To optimize simultaneous detection of these viruses, a new set of primers were designed from the coat protein region of the viruses to suit duplex reverse transcription polymerase chain reaction (D-RT-PCR) and the conditions were standardized to amplify the target viruses in this assay. Of the 29 sugarcane samples tested for the simultaneous occurrence of SCMV and SCSMV, 10 sugarcane varieties were found to be infected with both the viruses with positive amplifications of ∼860 bp and ∼690 bp respectively, for SCMV and SCSMV. In the remaining 19 varieties, SCSMV was detected alone as the causative virus of mosaic. None of the selected varieties were found infected with SCMV alone. Further, the D-RT-PCR was found equally reliable to uniplex RT-PCR performed with SCMV and SCSMV primers in separate reactions. The D-RT-PCR products were cloned, sequenced and the respective sequence information confirmed the authenticity of the viruses amplified in D-RT-PCR. The lengths of the amplified fragments from SCMV isolates were ranged between 812 and 877 bp and in SCSMV it ranged from 683 to 689 bp. This diagnostic approach will be useful to establish distribution map of the virus(es) associated with mosaic in India and in other sugarcane growing countries.

Real time RT-PCR with a newly designed set of primers confirmed the presence of 2b and 2x/d myosin heavy chain mRNAs in the rat slow soleus muscle

In order to re-evaluate the presence and relative quantity of 2b and 2x/d myosin heavy chain (MyHC) transcripts in rat slow soleus muscle by using real time RT-PCR we have compared the available relevant cDNA sequences and designed a new set of primers having similar melting temperatures, matching separate MyHC exons in the regions of maximal differences in MyHC coding sequences, and containing G or C at the 3 -end. These also yielded PCR products of corresponding length, which is an important requirement for real time RT-PCR quantification. The experiments were performed on 8-month-old inbred female Lewis strain rats used in our current study of regenerating transplanted muscles. The real time RT-PCR measurement confirmed the expression of all four MyHC mRNAs (type 1, 2a, 2x/d and 2b) in both fast extensor digitorum longus and slow soleus muscles, although in the soleus muscle of adult rats, only type 1 and 2a protein isoforms can be usually detected.

An epitope-specific PCR test for diagnosis of Leishmania donovani infections

The carboxy-terminal region of recombinant heat shock protein 70 of Leishmania donovani has been shown to have an immunodominant epitope. We designed a PCR assay using a new set of primers encompassing the gene sequence from 457bp to 927bp, which amplified a segment of the L. donovani genome in a species-specific manner. The assay was sensitive enough to detect 0.5pg of parasite DNA, which increased 10-fold (0.05pg) when an internal probe (583-609bp) was used in the Southern blot. The assay was able to detect parasite DNA from the liver and spleen of L. donovani-infected hamsters as well as lesion aspirates from parasitologically confirmed post-kala-azar dermal leishmaniasis (PKDL) and bone marrow aspirates from visceral leishmaniasis (VL) patients. No amplification was seen with axenically cultured promastigotes of L. infantum, L. tropica, L. major, L. mexicana, L. aethiopica or other intracellular organisms such as Entamoeba histolytica, Mycobacterium tuberculosis, Plasmodium vivax or human peripheral blood mononuclear cells. This PCR provides a specific tool for the diagnosis of VL and PKDL with simultaneous species identification.

Development of a new diagnostic marker for growth habit selection in faba bean (Vicia faba L.) breeding

Faba bean varieties with determinacy of the apical meristem are relevant to green production. A diagnostic CAPS (cleavage amplification polymorphic sequence) marker for determinate growth habit (ti) in faba bean was previously developed by Avila et al. (Mol Breed 17:185-190, 2006) but was effective only on a limited range of cultivars or genotypes. In this study, we studied the reasons for this limited application and developed a new marker useful for most faba bean-breeding programs. By designing a new set of primers, the complete genomic Vf_TFL1 sequences from different genotypes contrasting for the character were obtained and additional base changes associated with the ti phenotype were identified. The comparison among faba bean sequences showed that the previous CAPS marker was based on a SNP (single nucleotide polymorphism) at position 469 in the intron 2-3, a silent mutation. On the contrary, a SNP at position 26 that distinguishes determinate and indeterminate growth habit genotypes lead to an amino acid change (Leu-9 to Arg) in the determinate growth habit genotypes that could account for the ti phenotype. A dCAPS marker based on this SNP that creates a TaqI site in the ti allele was developed. The marker was 100% successful in predicting ti phenotypes in a broad range of faba bean germplasm representing all major cultivars historically grown in Europe. The outcome confirms the utility of the new dCAPS in worldwide marker-assisted selection programs.

Evaluation of a real-time PCR assay based on the repetitive B1 gene for the detection of Toxoplasma gondii in human peripheral blood

In this paper, we examined the diagnostic value of a real-time polymerase chain reaction (PCR) using fluorescence resonance energy transfer (TaqMan assay) with a new set of primers and probe targeting the B1 gene to reproducibly detect and quantify Toxoplasma gondii in human blood. A total of 183 buffy coat samples from patients serologically classified as recent toxoplasmosis (immunoglobulin M (IgM)+, n = 35) or chronic infection (IgM- and immunoglobulin G (IgG)+, n = 110), and seronegative individuals (n = 38) was investigated. Of the IgM seropositive patients, 17:35 (48.6%) presented parasitaemia, whereas 3.6% positivity was achieved in those individuals that theoretically corresponded to chronic infection (4:110). In the seronegative group, the assay provided 7.9% (3/38) of positive results. Interestingly, one of them was confirmed as positive in a conventional PCR targeting the Toxoplasma B1 gene after hybridization with an internal probe. Real-time PCR was able to accurately quantify the parasite load when concentrations of T. gondii DNA are low, revealing a parasite burden ranged from 9.92 x 10(-3) to 8.73 x 10(-1) tachyzoites genome per milliliter of blood. The chance of an IgM+ patient to present parasitemia detected by the TaqMan procedure was 19.02 times greater than in IgM- individuals (P < 0.05). It was observed a positive association between the optical density values of the IgM serological tests and the number of circulating parasites in the acute patients (P < 0.0001). The specificity of the molecular test was 95.3% when calculated using IgM+ patients as disease group and IgM- as nondisease group. The low sensitivity observed in the IgM seropositive group (48.6%) could be due to the use of buffy coat as clinical material for DNA extraction. An amplification control based on the human beta-actin gene was used in parallel to monitor PCR inhibition and to control for DNA integrity.

Development of a CAPS marker system for genotyping European pear cultivars harboring 17 S alleles

The full-length cDNAs of eight S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars that could not be characterized by the cleaved amplified polymorphic sequences (CAPS) marker system for genotyping European pear cultivars harboring nine S alleles Sa, Sb, Sd, Se, Sh, Sk, Sl, Sq, and Sr. Comparison of the nucleotide sequences between these cDNAs and six putative S-RNase alleles previously amplified by genomic PCR revealed that five corresponded to the putative Sc-, Si-, Sm-, Sn-, and Sp-RNase alleles and the other three corresponded new S-RNase alleles (designated as putative Sg-, Ss-, and St-RNase alleles). Genomic PCR with a new set of primers was used to amplify 17 S-RNase alleles: 1906 bp (Sg), 1642 bp (St), 1414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb), and ca. 350 bp (Sa, Sc, Sd, Sh, Si, Sm, Sn, Sp, Sr, and Ss). Among them, S-RNase alleles of similar size were discriminated by digestion with 11 restriction endo-nucleases. The PCR amplification of 17 S-RNase alleles following digestion with the restriction endonucleases provided a new CAPS marker system for rapid S-genotyping of European pear cultivars harboring 17 S alleles. Using the CAPS analysis, Sc, Sg, Si, Sm, Sn, Sp, Ss, and St alleles were found in 32 cultivars, which were classified into 23 S-genotypes.

Improving Access to the Control Region and tRNA Gene Clusters of Dipteran Mitochondrial DNA

Despite the successful use of universal primers for amplifying insect mtDNA, specific regions remain difficult to recover and demand the use of taxon-specific primers. In this work, we describe a new set of primers for efficiently amplifying and sequencing the mtDNA control region and three tRNA gene clusters of dipterans of medical and veterinary importance, including Muscidae, Calliphoridae, and Oestridae species. These new primers were useful for investigating the nucleotide information and the structural organization of dipteran mtDNA.

Corrigendum

The Plant JournalVolume 45, Issue 5 p. 869-869 Free Access Corrigendum First published: 07 February 2006 https://doi.org/10.1111/j.1365-313X.2006.02707.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume 40, Number 2, October 2004, pages 322–331, Ryu et al. The authors would like to make clear the following: The real-time quantitative RT-PCR results shown in Figures 3(c) and 4(b) were exaggerated. For RT-PCR analyses we had previously amplified fragments greater than 300 bp. It is suggested (see manufacturers’ protocol) that while using SYBR green for real time RT-PCR, the gene fragments to be amplified should be 50–150 bps (ABI User Bulletin no.2) so as to minimize the artifacts generated from non-specific amplicons and primer-dimers which results in an overestimation of the target concentration. We repeated these experiments with the primers designed to amplify fragments that are between 50 to100 bp using Primer Express® Software Version 2.0 (ABI). The fold differences of the silenced transcripts were drastically reduced when new set of primers was used. However, the trend of gene silencing remained the same and therefore the new data will not alter the overall conclusion of the manuscript. We apologise to our readers for this mistake. Reference Ryu, C.M., Anand, A., Kang, L., Mysore, K.S. (2004) Agrodrench: a novel and effective agroinoculation method for virus-induced silencing in roots and diverse Solanaceous species. The Plant Journal, 40, 322– 331. Volume45, Issue5March 2006Pages 869-869 ReferencesRelatedInformation

Integrated Genotyping and mRNA Expression Profiling of Killer Immunoglobulin-Like Receptors.

Natural killer (NK) cells belong to a subgroup of lymphocytes (CD3-CD56+) which play an important role in the cellular immune response against virus-infected cells and tumors. The activity of NK cells is regulated by a balance of triggering and inhibitory receptors, including Killer Ig-like Receptor (KIR) molecules which interact with specific HLA class I molecules, predominantly HLA-C, on target cells. The 17 known KIR genes are divided into two classes: activating KIRs and inhibitory KIRs. There is strong evidence that inhibitory KIR mismatch between donor and recipient improves the outcome of haploidentical hematopoietic stem cell transplantation (HSTC) in leukemia patients (Ruggeri et al. 2002). In addition, the KIR-HLA constellation is assumed to have an influence on the severity of graft versus host disease (GvHD). Whether these activities of NK cells are clinically important and to what extent these processes are mediated only by KIR-HLA class I interactions remains to be determined. In human populations, KIR gene haplotypes vary in the number and type of KIR genes they contain. Further diversification is observed by expanded allelic polymorphism at the individual genes. In general, KIR haplotypes contain 7–12 genes plus 2 pseudogenes. Extra KIR heterogeneity is provided at the expression level: different subsets of NK cells within an individual express different KIRs. Recently, it was shown that KIR genotyping alone does not seem to be sufficient for donor KIR assessment because of the lack of gene expression in approximately one-fourth of the individuals for one of the inhibitory KIRs that recognize the three major groups of MHC class I ligands (Leung et al. 2005). KIR phenotyping by flow cytometry using monoclonal antibodies is insufficient due to the lack of specific monoclonal antibodies. For trustworthy analysis, one has to combine KIR genotyping with mRNA expression profiling and flow cytometry. Therefore, we developed a new set of sequence-specific primers (SSP). This primer set can be applied to perform either KIR genotyping or mRNA expression profiling despite the high degree of identity of the genes (80–90%, sometimes more than 95%). The primers of each KIR gene (15 genes and 2 pseudogenes) cover all allelic variants annotated by the IPD KIR Sequence Data Base (status quo July 05). Using this primer set, we genotyped 25 individuals, and compared the results with other sets of KIR primers published elsewhere. Additionally, we show the mRNA expression profile employing the same set of new primers. We confirmed these results on the protein level by flow cytometry.

Mutation analysis of the HFE gene associated with hereditary hemochromatosis in a Venezuelan sample

The frequency of the C282Y, H63D and S65C alleles of the HFE gene was determined in a sample of the Venezuelan population. Two new sets of primers were tested for amplifying the regions mapping these mutations, and genotyping was performed by restriction fragment length polymorphism (RFLP). DNA sequencing was used to validate the RFLP analysis. Serum ferritin levels were also determined. Two hundred and fourteen individuals were tested, extracting DNA from whole blood cells (n=177) or from serum (n=37). The frequency of heterozygous subjects was 3.7, 18.2 and 1.7% for the C282Y, H63D and S65C mutations, respectively, and the allele frequencies were 0.019+/-0.01 for C282Y, 0.119+/-0.016 for H63D and 0.009+/-0.005 for S65C. The results suggest that the admixture of native populations with subjects of South European origin might have had an important role in the diffusion of HFE alleles in Venezuela. C282Y homozygous subjects were not found in this study. No HFE genotype studied here was associated with a significant elevation of serum ferritin concentrations, except for C282Y/H63D compound heterozygote found in one asymptomatic male. This finding supports the theory that the H63D mutation could be involved in alterations of iron parameters when inherited together with C282Y. Our results indicate that C282Y homozygotes will be rarely detected. Performance of HFE mutation analysis in individuals with high iron determinations would be recommended.

Conservation and versatility of a new set of primers for long‐PCR amplification of complete insect mitochondrial genomes based on Haematobia irritans mtDNA sequences

AbstractThe amplification of complete mitochondrial genomes by long PCR (polymerase chain reaction) has been a major contribution to the large‐scale sequencing of arthropodan mitochondrial genomes. In this work, we designed six conserved long‐PCR primers to successfully recover the entire mitochondrial genome of the horn fly Haematobia irritans (Diptera: Muscidae) in two overlapping fragments. The conservation and versatility of these primers were tested for 17 other species from four major insect orders: Diptera (14), Coleoptera (1), Lepidoptera (1) and Hymenoptera (1). The amplification of complete mitochondrial genomes in orders other than Diptera suggested an even broader application of these primers, especially within the Hexapoda.

Characterization of a New Molecular Marker for Investigating Skate Population Genetics: Analysis of Three Mediterranean Skate Species (genusRaja) of Commercial Interest as a Test Case

In this study we describe a new set of primers specifically designed for the amplification of the mitochondrial control region in skates of the Rajinae subfamily. The suitability of the amplified region as molecular marker was tested on three Mediterranean skates species of commercial interest (Raja clavata, Raja asterias and Raja miraletus). Fifty-six skate samples collected from 6 locations around the Italian peninsula were molecularly typed for 324–332bp of the mitochondrial DNA control region. The target sequence was polymorphic and, therefore, informative in all three species. Although the number of individuals screened for each species/location was small, our data suggest that populations of R. clavata and R. miraletus in central Mediterranean are single stocks. Conversely, there may be two genetically differentiated forms of R. asterias: one in the Adriatic Sea and the other in the Ligurian/Tyrrehenian Seas which represent separate geographic units or stocks. The highest level of genetic variability was recorded for R. clavata (H = 0.61) and the lowest for R. miraletus (H = 0.17), with R. asterias showing an intermediate level (H = 0.29), though no variation was found within the two (Adriatic and Ligurian/Tyrrehenian) sub-populations of the latter. These preliminary data show the potential of the described primers set to be employed in skate population genetics and phylogeny.

Parvicapsula pseudobranchicola (Myxosporea) in farmed Atlantic salmon Salmo salar: tissue distribution, diagnosis and phylogeny

Parvicapsula pseudobranchicola infections in farmed Atlantic salmon in Norway are associated with low-grade to significant mortalities. The parasite is found as mature spores in pseudobranchs, but has also been detected in the gills, liver and kidney. Diagnosis has relied on the detection of Parvicapsula spores, with the pseudobranch being the preferred organ. A better understanding of the epizootiology of this myxosporean is a prerequisite for appropriate management and control. Hence, early detection of infections and life cycle studies are needed. We sequenced the small subunit (ssu) rDNA (18S) from P. pseudobranchicola and developed a sensitive diagnostic PCR protocol. This allowed us to (1) identify appropriate tissues for diagnostic assays, (2) examine the intraspecific variation in ssu rDNA in the parasite's Norwegian range, (3) examine annelid potential primary hosts and (4) obtain additional ssu rDNA sequences of marine Parvicapsula species to perform a phylogenetic study. Primers were constructed targeting the ssu rDNA from P. minibicornis. With these we obtained a partial ssu sequence of the P. pseudobranchicola type isolate. A new set of primers (PCF3/PCR3) was constructed for diagnostic purposes. These were tested against DNA from the host and several myxozoan species infecting Norwegian salmon. The primers give a positive product of 203 bp and pick out P. pseudobranchicola in salmnonids. They also amplify the congeners P. unicornis and P. asymmetrica infecting unrelated fish. The PCR protocol developed showed a greater sensitivity than light microscopy. The pseudobranchs were always positive and are the recommended organ for PCR diagnostics. There was no sequence variation between geographic isolates from farmed salmon. Preliminary examinations of marine polychaetes and oligochaetes collected from farm sites with parvicapsulose problems were negative. A comparison of the sequence of the ssu rDNA from P. pseudobranchicola with that of other myxozoans shows that it groups closely together with P. unicornis and P. asymmetrica. The closest relative to this group is P. minibicornis.

Evolutionary conservation and versatility of a new set of primers for amplifying the ribosomal internal transcribed spacer regions in insects and other invertebrates

AbstractThe evolutionary conservation and versatility of a new set of nuclear primers for the amplification of the ribosomal internal transcribed spacer regions in insects and other invertebrates have been studied. These primers, conserved across Insecta and other invertebrates, are based on a comprehensive taxonomic survey of the current DNA sequence databases. Their versatility was demonstrated by extensive polymerase chain reaction assays in 16 species from two arachnid orders, eight insect orders, three invertebrate and vertebrate chordate orders and by direct sequencing of the amplified products. The availability of these primers to the insect research community should facilitate the use of internal transcribed spacer regions in intraspecific studies as well as phylogenetic analysis of closely related taxa.

Cloning and sequence analysis of a gene encoding polygalacturonase-inhibiting protein from cotton*

Abstract Polygalacturonase-inhibiting proteins PGIP play important roles in plant defense of pathogen especially fungi. A pair of degenerated primers is designed based on the conserved sequence of 20 other known pgip genes and used to amplify Gossypium bar-badense cultivation 7124 cDNA library by touch-down PCR. A 561 bp internal fragment of the pgip gene is obtained and used to design the primers for rapid amplification of cDNA ends. A composite pgip gene sequence is constructed from the products of 5′ and 3′ RACE which are 666 bp and 906 bp respectively. Analysis of nucleic acid sequence shows 69.2% and 68.7% similarity to Citrus and Poncirus pgip genes respectively. Its open reading frame of the gene encodes a polypeptide of 330 amino acids in which 10 leucine-rich repeats arrange tandemly. A new set of primers is designed to the 5′ and 3′ ends of the gene which allows amplification of the full-length gene from the cotton cDNA library. Genomic DNA analysis reveals that this gene has no intron.