Designing degenerate pcr primers genome biology full text. Accordingly, degenerate primers that represented all possible nucleotides found in the respective potyvirus sequences were synthesized. Genefisher2 is a webbased program that helps user to find primers for degenerated sequences. Annealing temperature ideally 44c degeneracy how many variations of the ad primer there are ideally 128 or 256. Apr 27, 2000 the purpose of codehop is to help in the design of degenerate pcr primers from protein sequence. Detection of potyviruses infecting iranian common beans. Two pairs of degenerate primers were designed from sequences within the potyviral ci ciforcirev and hcprocoding regions.
Potyvirus detection facilitated by degenerate primers 1. Multiplex degenerate primer design for targeted whole genome. 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. 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. Background kemia virus mmulv reverse transcriptase fermentas, lithuania was used in the synthesis as described. Therefore to identify rna virus species, degenerate primers i. Degenerate primers facilitate the detection and identification of potyviruses from. Mark an included region to pick primers fixed at its the boundaries. The nib primers detected all 40 potyvirus isolates, but only 80 and 50% of isolates were detected with the wcien and potyvirid primers respectively.
This work is part of my phd program which was supported by the. It corresponds to the primers aacggag, aacggcg, aacgggg, aactgag, aactgcg, aactggg. Nov 11, 20 by designing primers using degenerate positions all the possible variants of the target sequence can be amplified. Molecular evidence for a new potyvirus species in yam. Cassava is the main staple food for over 800 million people globally. 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. 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. Pdf degenerate primers facilitate the detection and. Comparison of genusspecific primers in rtpcr for the. Characterization of a potyvirus associated with yellow mosaic. The potyvirus genus is one of the largest genera of plant viruses and encompasses many economically important pathogens. A phylogenetic tree produced by neighboring join nj method. Consensus backtranslation, codon table backtranslation, dna and primer. Rapid and sensitive detection of potyvirus infecting.
A primer for designing degenerate primers bitesize bio. September 2008 registration form and license agreement. Biological and molecular characterization of potato virus y. The software incorporates batcons2 which allows to calculate ambiguous consensus sequence and backtranslation of aminoacid to nucleicacidsequences. Designing degenerate pcr primers for templates of unknown nucleotide sequence may be a very difficult task. Degenerate primers are a mixture of similar, but not identical primers that are used to amplify a same gene from different organisms. I have to align around 1290 nucleotide sequences available in the genbank and then design primers based on the consensus seen. Programme for degenerate primer design bioinformatics and.
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. Pdf a novel pair of universal primers for the detection of. A molecule, which may be a small polymer, that initiates the synthesis of a larger structure. Degenerate primers cichlid labs university of maryland. A novel pair of universal primers for the detection of.
For example, aacg,tga,c,gg is a 7long degenerate primer, in which the fourth and sixth positions are degenerate. 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. Reverse table 4 sequence of degenerate primers for subgroup detection in rtpcr, sense and antisense speciesspeci. The identity of 12 unknown potyvirus isolates was con. A pheromone that causes a longterm physiologic change. Jun 19, 2007 in an attempt to characterize those potential yam potyviruses, total rna was extracted from one d. Pdf design and application of two novel degenerate primer pairs. 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. Alternatively, degenerate primers are used when primer design is based on protein sequence, as several different codons can code for one amino acid. Accumulating variation at conserved sites in potyvirus. Accumulating variation at conserved sites in potyvirus genomes is. Pdf two pairs of degenerate primers were designed from sequences within the.
This general potexvirus primer set can be useful for the identification of unknown potexvirus infections. Hyden a software for designing degenerate primers my. Degenerate primers design tools for qpcr are able to calculate and generate primer sequences using specific. Degenerate primers a primer is degenerate if some of its positions admit more than one nucleotide. 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. 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. Identification of potyviruses using the polymerase chain. 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. Using the cifor and cirev primers, three novel potyviruses infecting crop and weed species from vietnam were detected, namely telosma mosaic virus telmv infecting. Hyden highly degenerate primers is a program for designing pairs of degenerate primers for a given set of dna sequences. The degenerate primers for the genus potyvirus direct the amplification of highly conserved 700bp or 1600bp fragments from the 3 terminus of most potyviruses. Hyden registration a software for designing degenerate primers.
The nib primer pair was used to amplify dna from two virus isolates, wvmv and an unknown potyvirus from g. In an attempt to characterize those potential yam potyviruses, total rna was extracted from one d. 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. Detection of an unidentified potyvirus from roystonea regia. Design and application of two novel degenerate primer. 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. The use of pcr for cloning of large cdna fragments of turnip. 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. Rtpcr analysis of the total rna isolated from different potyvirus infected tuber crops resulted in the amplification of 327 bp amplicon figure 1. Jul 10, 2008 degenerate pcr involves using primers that allow for some wiggle room in the sequence of the primers. Molecular characterization of tuberose mild mosaic virus and. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. Rapid and sensitive detection of potyvirus infecting tropical. Unusual occurrence of a dag motif in the ipomovirus cassava.
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. Overview hyden highly degenerate primers is a program for designing pairs of degenerate primers for a given set of dna sequences. 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. 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. Potyviridae as a major challenge to growing cucurbits in. These degenerate primers were paired with an oligodt primer that annealed to the polyadenylate tail that is present at 3. Chaim linhart and ron shamir telaviv university original version. Design arbitrary degenerate primers with nonbinding criteria. Optionally targets and includedexcluded regions can be specified.
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. Pdf a novel pair of universal primers for the detection. 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. With this technique, unambiguous species allocation was achieved for 34 potyvirus.
Degenerate primer definition of degenerate primer by. Cassava brown streak virus cbsv and ugandan cassava brown streak virus ucbsv, with up to 100% yield loss for smallholder farmers in the region. Rna with potyvirus specific degenerate and tmmv coat protein specific primers by reversetranscription polymerase chain reaction rtpcr. Potyvirus detection facilitated by degenerate primers 116 iran j biotech. Hello, i am looking at designing degenerate primers to detect between genotypes of viruses. Select primer pairs to detect the given template sequence. Pcrdetection polymerase potexvirus virus identification this. Specificity of potyvirus degenerate primers total rna were extracted from each virusinfected plant and analyzed by rtpcr with potyvirus degenerate primers, mj1 and mj2. Several sets of degenerate primers exist that detect conserved regions of potyvirus genomes through rtpcr tests ha et al. Rtpcr was conducted with potyvirus degenerate primers and the amplicons were sequenced to identify more specifically the potyviruses. Development of a general potexvirus detection method. A novel pair of universal primers for the detection of potyviruses. 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. Empetrum cbf gene were isolated by pcr with degenerate oligonucleotide primers 5.
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. Dna was amplified using potyvirus specific degenerate primers and reverse transcriptase pcr. Potyvirus detection facilitated by degenerate primers. 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. All four viruses shared genomic features typical of potyviruses. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Jf3 was a 20mer oligonucleotide with a 32fold degeneracy and was entirely deduced from the nib homology region. Hp plant dna mini kit omega biotek was utilized for. Design and application of two novel degenerate primer pairs for the. The denatured purified virus had single major polypeptide of molecular weight 32 kda. I would like to design a number of arbitrary degenerate primers primers with variable bases, e. Hyden works well for large input sets of genomic sequences e.
Two pairs of degenerate primers were designed from sequences within the potyviral ci ciforcirev and hcprocoding regions hpfohprev. Its production in eastern africa is being constrained by two devastating ipomoviruses that cause cassava brown streak disease cbsd. 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. The disease was transmitted to tomatillo plants by the green peach aphid myzus persicae in a nonpersistent manner. It includes 111 confirmed species and 86 tentative species 3. Rtpcr amplifications with universal primers have facilitated the detection of. 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. A degenerate primer pair was designed to amplify a. 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. Overlapping potexvirus cdna clones were generated using degenerate genus potexvirus replicase primers and, later, virusspecific primers in 3 race. A potyvirus potato virus y, pvy causing severe mosaic mottling in potato solanum tuberosum was identified.
I would like to be able to specify the following parameters for the design function. For the cheapest degenerate primers, make the first base one single base. Degenerate primers are just a mix of primers with similar sequences. Design and application of two novel degenerate primer pairs. The pcr products were cloned, sequenced and analysed and contained a 5. Coexistence of three virus genera badnavirus, potyvirus. Degenerate primer design bioinformatics tools omicx. Degenerate primers facilitate the detection and identification of. First, by the use of genedoc software 26, sequences of the new isolates.
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