Design and application of two novel degenerate primer. Jul 10, 2008 degenerate pcr involves using primers that allow for some wiggle room in the sequence of the primers. Select primer pairs to detect the given template sequence. Nov 11, 20 by designing primers using degenerate positions all the possible variants of the target sequence can be amplified. Using the cifor and cirev primers, three novel potyviruses infecting crop and weed species from vietnam were detected, namely telosma mosaic virus telmv infecting telosma. This general potexvirus primer set can be useful for the identification of unknown potexvirus infections. Hyden registration a software for designing degenerate primers. Background kemia virus mmulv reverse transcriptase fermentas, lithuania was used in the synthesis as described. Degenerate primers are a mixture of similar, but not identical primers that are used to amplify a same gene from different organisms. I would like to be able to specify the following parameters for the design function. Degenerate pcr is a pcr method that uses degenerate primers to amplify unknown dna sequences that are related to a known dna sequence or to amplify a mixture of related sequences in one pcr reaction.
A pheromone that causes a longterm physiologic change. Hyden works well for large input sets of genomic sequences e. Design and application of two novel degenerate primer pairs. Biological and molecular characterization of potato virus y. Programme for degenerate primer design bioinformatics and. The nib primer pair was used to amplify dna from two virus isolates, wvmv and an unknown potyvirus from g. I have to align around 1290 nucleotide sequences available in the genbank and then design primers based on the consensus seen.
A novel pair of universal primers for the detection of. Pdf a novel pair of universal primers for the detection of. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Using the cifor and cirev primers, three novel potyviruses infecting crop and weed species from vietnam were detected, namely telosma mosaic virus telmv infecting. Mark an included region to pick primers fixed at its the boundaries. Degenerate primer definition of degenerate primer by. Hello, i am looking at designing degenerate primers to detect between genotypes of viruses. In an attempt to characterize those potential yam potyviruses, total rna was extracted from one d. Detection of potyviruses infecting iranian common beans. Degenerate primers design tools for qpcr are able to calculate and generate primer sequences using specific. A phylogenetic tree produced by neighboring join nj method.
The most 3 base could be degenerate, but it is quite expensive to have degenerate primers made where the first base the most 3 is degenerate. Rtpcr was conducted with potyvirus degenerate primers and the amplicons were sequenced to identify more specifically the potyviruses. Sample sequences were highly diverse table 1, confirming the biological variability observed in the greenhouse assays. Two pairs of degenerate primers were designed from sequences within the potyviral ci ciforcirev and hcprocoding regions hpfohprev, and these were shown to be highly specific to. A mix of oligonucleotide sequences in which some positions contain a number of possible bases, giving a population of primers with similar sequences that cover all possible nucleotide combinations for a given protein sequence iserte 20. Two pairs of degenerate primers were designed from sequences within the potyviral ci ciforcirev and hcprocoding regions hpfohprev. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. Cassava brown streak virus cbsv and ugandan cassava brown streak virus ucbsv, with up to 100% yield loss for smallholder farmers in the region. With this technique, unambiguous species allocation was achieved for 34 potyvirus.
Hyden a software for designing degenerate primers my. Identification of potyviruses using the polymerase chain. Pdf two pairs of degenerate primers were designed from sequences within the. Degenerate primer design bioinformatics tools omicx. The aim of this study to characterize this potyvirus on the basis of biological, serological and partial nucleotide sequence properties from different locations of rajasthan. Jun 19, 2007 in an attempt to characterize those potential yam potyviruses, total rna was extracted from one d. Design arbitrary degenerate primers with nonbinding criteria. A virus isolate associated with yellow mosaic disease was purified from commercially cultivated jasmine jasminum sambac from andhra pradesh, india and it contained flexuous filamentous particles of 720. Consensus backtranslation, codon table backtranslation, dna and primer. All four viruses shared genomic features typical of potyviruses. Degenerate primers a primer is degenerate if some of its positions admit more than one nucleotide. Identification of potyviruses infecting vanilla by direct.
In this paper, we present a new method to design degenerate primers, implemented in familyspecific degenerate primer design fasdpd computer software, for which the starting point is a multiple alignment of related amino acids or nucleotide sequences. The potyvirus genus is one of the largest genera of plant viruses and encompasses many economically important pathogens. Characterization of a potyvirus associated with yellow mosaic. Degenerate primers are just a mix of primers with similar sequences.
I would like to design a number of arbitrary degenerate primers primers with variable bases, e. Molecular evidence for a new potyvirus species in yam. Two pairs of degenerate primers were designed from sequences within the potyviral ci ciforcirev and hcprocoding regions. Two pairs of degenerate primers were designed from sequences within the potyviral ci ciforcirev and hcprocoding regions hpfohprev, and these were shown to be highly specific to members of the genus potyvirus. Rtpcr amplifications with universal primers have facilitated the detection of. Pcr assay with degenerate primers followed by direct sequencing of a 327 bp coat protein gene fragment was used to identify the potyviruses infecting vanilla. Pdf degenerate primers facilitate the detection and. Potyvirus detection facilitated by degenerate primers. Background the genus potyvirus in the family potyviridae is one of the largest plant virus genera 1 that are accounted for 40% of losses caused by all plant viruses 2.
Hyden highly degenerate primers is a program for designing pairs of degenerate primers for a given set of dna sequences. Pdf design and application of two novel degenerate primer pairs. Designing degenerate primers degenerate primers may be used to amplify dna in situations where only the protein sequence of a gene is known, or where the aim is to isolate similar genes from a variety of species. Jf3 was a 20mer oligonucleotide with a 32fold degeneracy and was entirely deduced from the nib homology region. Degenerate primers allow for the pcr amplification of a wider range of viral isolates with only one set of premixed primers, thus increasing amplification success. Degenerate primers facilitate the detection and identification of. Overlapping potexvirus cdna clones were generated using degenerate genus potexvirus replicase primers and, later, virusspecific primers in 3 race. Rna with potyvirus specific degenerate and tmmv coat protein specific primers by reversetranscription polymerase chain reaction rtpcr. For example, aacg,tga,c,gg is a 7long degenerate primer, in which the fourth and sixth positions are degenerate. Accumulating variation at conserved sites in potyvirus genomes is. For the cheapest degenerate primers, make the first base one single base. Rapid and sensitive detection of potyvirus infecting tropical.
It includes 111 confirmed species and 86 tentative species 3. Chaim linhart and ron shamir telaviv university original version. A primer for designing degenerate primers bitesize bio. Pcrdetection polymerase potexvirus virus identification this. Two pairs of degenerate primers, designed in the ci gene ciforcirev and hcpro gene hpfohprev, were developed for the detection of viruses in the genus potyvirus. The identity of 12 unknown potyvirus isolates was con. A potyvirus potato virus y, pvy causing severe mosaic mottling in potato solanum tuberosum was identified. In a highthroughput environment, to pcr amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate pcr primers is an important strategy. Coexistence of three virus genera badnavirus, potyvirus. Potyviridae as a major challenge to growing cucurbits in. Therefore, dna was amplified using potyvirusspecific degenerate primers and reverse transcriptase pcr. Given a multiple alignment of a series of target proteins, codehop will transform the aminoacid consensus sequence into a dna consensus sequence, using a userdesignated codonusage table. Apr 27, 2000 the purpose of codehop is to help in the design of degenerate pcr primers from protein sequence.
Molecular characterization of tuberose mild mosaic virus and. Annealing temperature ideally 44c degeneracy how many variations of the ad primer there are ideally 128 or 256. Potyvirus detection facilitated by degenerate primers 1. The software incorporates batcons2 which allows to calculate ambiguous consensus sequence and backtranslation of aminoacid to nucleicacidsequences. Therefore to identify rna virus species, degenerate primers i. A degenerate primer pair was designed to amplify a. Overview hyden highly degenerate primers is a program for designing pairs of degenerate primers for a given set of dna sequences. Implementation degeneracy is a critical factor in defining sensitivity in pcr since a highly degenerate primer will have a few matches that precisely match the template. The disease was transmitted to tomatillo plants by the green peach aphid myzus persicae in a nonpersistent manner. Degenerate primers facilitate the detection and identification of potyviruses from. Specificity of potyvirus degenerate primers total rna were extracted from each virusinfected plant and analyzed by rtpcr with potyvirus degenerate primers, mj1 and mj2. A novel pair of universal primers was developed to detect potyvirus species after conserved sites were identified using all fulllength potyvirus sequences available by 2005.
A novel pair of universal primers for the detection of potyviruses. Comparison of genusspecific primers in rtpcr for the. Designing degenerate pcr primers genome biology full text. The use of pcr for cloning of large cdna fragments of turnip. Degenerate primers cichlid labs university of maryland. Its production in eastern africa is being constrained by two devastating ipomoviruses that cause cassava brown streak disease cbsd. Designing degenerate pcr primers for templates of unknown nucleotide sequence may be a very difficult task. Sep 28, 2007 two pairs of degenerate primers were designed from sequences within the potyviral ci ciforcirev and hcprocoding regions hpfohprev, and these were shown to be highly specific to members of the genus potyvirus. To further identify the causal agent, total nucleic acids were extracted using a ctab method 2 and tested by reverse transcription rtpcr using potyvirus degenerate primers cifor and cirev 1. Design and application of two novel degenerate primer pairs for the. Dna was amplified using potyvirus specific degenerate primers and reverse transcriptase pcr.
Elsevier journal of virological methods 50 1994 211218 journal of virological methods detection of an unidentified potyvirus from roystonea regia palm using the polymerase chain reaction and degenerate, potyvirus specific, primers and potential problems arising from the amplification of host plant dna sequences m. It corresponds to the primers aacggag, aacggcg, aacgggg, aactgag, aactgcg, aactggg. Unusual occurrence of a dag motif in the ipomovirus cassava. While a number of degenerate primers for use in broad spectrum rtpcr assays have been published, it is not clear which of these primers would be the most useful for use by plant diagnostic laboratories. Potyvirus detection facilitated by degenerate primers 116 iran j biotech. In the same year, colinet and kummert reported the qmkaaa primer independently, and produced primers to the wkhwi and wngsl sites from the niapro and nib genes 12.
The pcr products were cloned, sequenced and analysed and contained a 5. The nib primers detected all 40 potyvirus isolates, but only 80 and 50% of isolates were detected with the wcien and potyvirid primers respectively. Rtpcr analysis of the total rna isolated from different potyvirus infected tuber crops resulted in the amplification of 327 bp amplicon figure 1. A molecule, which may be a small polymer, that initiates the synthesis of a larger structure. Hp plant dna mini kit omega biotek was utilized for. Pdf a novel pair of universal primers for the detection.
This work is part of my phd program which was supported by the. September 2008 registration form and license agreement. Genefisher2 is a webbased program that helps user to find primers for degenerated sequences. Reverse table 4 sequence of degenerate primers for subgroup detection in rtpcr, sense and antisense speciesspeci. The denatured purified virus had single major polypeptide of molecular weight 32 kda. First, by the use of genedoc software 26, sequences of the new isolates. Development of a general potexvirus detection method. For example the 4th nucleotide in the primer sequence may allow it to anneal to template sequence with nucleotides a, t, or g, while excluding those with c. Detection of an unidentified potyvirus from roystonea regia. Several sets of degenerate primers exist that detect conserved regions of potyvirus genomes through rtpcr tests ha et al. Rapid and sensitive detection of potyvirus infecting. These degenerate primers were paired with an oligodt primer that annealed to the polyadenylate tail that is present at 3.
Empetrum cbf gene were isolated by pcr with degenerate oligonucleotide primers 5. Accordingly, degenerate primers that represented all possible nucleotides found in the respective potyvirus sequences were synthesized. Optionally targets and includedexcluded regions can be specified. Multiplex degenerate primer design for targeted whole genome.
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