Identification of novel connexins by reduced-stringency hybridization and PCR amplification using degenerate primers

Gap Junctions ◽  
1993 ◽  
pp. 15-20 ◽  
Author(s):  
DAVID L. PAUL ◽  
ROBERTO BRUZZONE ◽  
JACQUES-ANTOINE HAEFLIGER
Keyword(s):  
Pcr Amplification ◽  
Degenerate Primers

scholarly journals PrimerPooler: automated primer pooling to prepare library for targeted sequencing

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Silas S. Brown ◽  
Yun-Wen Chen ◽  
Ming Wang ◽  
Alexandra Clipson ◽  
Eguzkine Ochoa ◽  
...  
Keyword(s):  
Variant Calling ◽  
Pcr Amplification ◽  
Illumina Miseq ◽  
Degenerate Primers ◽  
Cross Hybridization ◽  
Targeted Next Generation Sequencing ◽  
Target Sequencing ◽  
A Genome ◽  
Experimental Protocols ◽  
Minimal Depth

Abstract Targeted next-generation sequencing based on PCR amplification involves pooling of hundreds to thousands of primers, for preamplification and subsequent parallel single/multiplex PCR. It is often necessary to allocate the set of primers into subpools, a common issue being potential cross-hybridization. For smaller numbers of primers, pool division can be done manually using trial and error to minimize potential hybridization, but this becomes inefficient and time consuming with increasing numbers of primer pairs. We developed PrimerPooler that automates swapping of primer pairs between any user-defined number of subpools to obtain combinations with low-potential interactions. PrimerPooler performs inter-/intra-primer hybridization analysis to identify the adverse interactions, as well as simultaneous mapping of all primers onto a genome sequence in a single run without requiring a prior index of the genome. This allows detection of overlapping primer pairs and allocation of these primer pairs into separate subpools where tiling approaches are used. Using PrimerPooler, 1153 primer pairs were assigned to three preamplification pools (388, 389 and 376 primer pairs each), then 144 subpools of six- to nine-plex PCR for Fluidigm Access Array PCR, followed by Illumina MiSeq sequencing. With optimized experimental protocols, an average of 3269 reads was achieved for the targeted regions, with 95% of targets covered by at least 50 reads, the minimal depth of reads for confident variant calling. PrimerPooler provides a fast and highly efficient stratification of primer pairs for targeted enrichment, thus ensuring representative amplification of the targeted sequences. PrimerPooler is also able to analyse degenerate primers, and is thus also useful for microbiological identification and related target sequencing.

scholarly journals Bias in Template-to-Product Ratios in Multitemplate PCR

1998 ◽  
Vol 64 (10) ◽  
pp. 3724-3730 ◽  
Author(s):  
Martin F. Polz ◽  
Colleen M. Cavanaugh
Keyword(s):  
Bacterial Species ◽  
Gene Copy Number ◽  
Pcr Amplification ◽  
Binding Energies ◽  
Site Directed Mutagenesis ◽  
Universal Primers ◽  
Gene Copy ◽  
Degenerate Primers ◽  
Relative Product ◽  
Genomic Dnas

ABSTRACT Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.

scholarly journals Distribution and Diversity of Geminiviruses in Trinidad and Tobago

1998 ◽  
Vol 88 (12) ◽  
pp. 1262-1268 ◽  
Author(s):  
Pathmanathan Umaharan ◽  
Malla Padidam ◽  
Ralph H. Phelps ◽  
Roger N. Beachy ◽  
Claude M. Fauquet
Keyword(s):  
Trinidad And Tobago ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Sweet Pepper ◽  
Yellow Mosaic Virus ◽  
Restriction Enzyme Digestion ◽  
Weed Species ◽  
Dot Blot ◽  
Degenerate Primers

Seven crop and eight weed species from 12 agricultural locations in Trinidad and Tobago were assayed for the presence of whitefly-transmitted geminiviruses (WTGs) by using dot blot hybridization and polymerase chain reaction (PCR) amplification of the N-terminal coat protein sequence with degenerate primers. The amplified fragments were cloned and analyzed by restriction enzyme digestion to determine fragment length polymorphism among the cloned fragments. Representative clones were then sequenced and subjected to phylogenetic analysis to determine the sequence similarity to known WTGs. WTGs were found in every location sampled and in 10 of the 15 species investigated: Lycopersicon esculentum(tomato), Capsicum annuum (pepper), Capsicum frutescens (sweet pepper), Abelmoschus esculentus (okra), Phaseolus vulgaris (beans), Alternanthera tenella, Desmodium frutescens, Euphorbia heterophylla, Malva alceifolia, and Sida acuta. The geminiviruses infecting these plants were closely related to potato yellow mosaic virus from Venezuela (PYMV-VE) and tomato leaf curl virus from Panama (ToLCV-PA). However, in pepper, sweet pepper, okra, Alternanthera tenella, Euphorbia heterophylla, Des-modium frutescens, and in one sample of tomato, a PYMV-VE-related virus was found in mixed infections with a virus related to pepper huasteco virus. Full-length infectious DNA-A and DNA-B of a tomato-infecting geminivirus from Trinidad and Tobago were cloned and sequenced. DNA-A appears to be a recombinant derived from PYMV-VE or ToLCV-PA, and Sida golden mosaic from Honduras. The implications of these findings in the control of WTGs are discussed.

scholarly journals A Begomovirus Associated with Leaf Curling and Chlorosis of Soybean in Sinaloa, Mexico is Related to Pepper golden mosaic virus

Plant Disease ◽  
2006 ◽  
Vol 90 (1) ◽  
pp. 109-109 ◽  
Author(s):  
J. Méndez-Lozano ◽  
E. Quintero-Zamora ◽  
M. P. Barbosa-Jasso ◽  
N. E. Leyva-López ◽  
J. A. Garzón-Tiznado ◽  
...  
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Intergenic Region ◽  
Pcr Amplification ◽  
Nucleotide Identity ◽  
Homologous Region ◽  
Degenerate Primers ◽  
Sequence Comparisons ◽  
Leaf Curling ◽  
Cp Gene

Since June 2001, symptoms of yellowing, leaf curling, crumpling, and stunted growth were observed on soybean (Glycine max Merr.) plants in Sinaloa, Mexico. These symptoms and the presence of whiteflies (Bemisia tabaci Gennadius) in the affected fields suggested a viral etiology. Samples from symptomatic plants were collected from commercial fields and analyzed for the presence of begomoviruses using DNA hybridization, and as a probe, the DNA A of Pepper huasteco virus at low stringency (2). Thirty-five positive samples were subsequently used for polymerase chain reaction (PCR) amplification with the degenerate primers RepMot and CPMot (1). These primers direct the amplification of a DNA A segment comprising the entire intergenic region (IR) and the first 210 bp of the coat protein (CP) gene, which is highly variable in size and nucleotide sequence among begomoviruses. PCR products were obtained for 25 of 35 samples and five of these were cloned into the pGEM-T easy vector (Promega, Madison, WI) and sequenced. The 571-bp DNA sequence (GenBank Accession No. AY905553) was compared with sequences of other begomoviruses in GenBank using the Clustal alignment method (MegAlign, DNASTAR software, London). The sequence was 74 and 70% identical to the Pepper golden mosaic virus (PepGMV; GenBank Accession No. U57457) and Cabbage leaf curl virus (CaLCuV; GenBank Accession No. U65529) sequences, respectively. Interestingly, the partial coat protein gene sequence (210 nt) of this soybean-infecting virus was 98% identical to the CP gene of Tobacco apical stunt virus (TbASV; GenBank Accession No. AF076855). Nonetheless, the known sequence of TbASV intergenic region (GenBank Accession No. AF077744) is very different from the homologous region of the soybean virus (34% of nucleotide identity). Analysis of the soybean virus intergenic region revealed that it harbors almost identical iterons (i.e., Rep-binding sites) to PepGMV, suggesting a close relationship between these two viruses. Soybean-infecting geminiviruses have been previously reported only from Asia; however, the partial sequence of a begomovirus isolated from soybean in Brazil was recently deposited in Genbank (Accession No. AY436328). Sequence comparisons between the Brazilian and Mexican isolates showed these viruses are less related with a nucleotide identity of 46%. Taken together, our data indicate that the virus identified in this study might be either a different strain of PepGMV adapted to leguminous plants or a new begomovirus species. To our knowledge, this is the first report of a begomovirus infecting soybean in Mexico. References: (1) J. T. Ascencio-Ibañez et al. Plant Dis. 86:692, 2002. (2) J. Méndez-Lozano et al. Phytopathology 93:270, 2003.

scholarly journals Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean

2005 ◽  
Vol 71 (12) ◽  
pp. 7910-7919 ◽  
Author(s):  
Rebecca J. Langlois ◽  
Julie LaRoche ◽  
Philipp A. Raab
Keyword(s):  
Pacific Ocean ◽  
Atlantic Ocean ◽  
Heterotrophic Bacteria ◽  
Pcr Amplification ◽  
Environmental Dna ◽  
Degenerate Primers ◽  
Community Shift ◽  
The Pacific ◽  
Group A ◽  
The Pacific Ocean

ABSTRACT To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.

scholarly journals Two Distinct Monooxygenases for Alkane Oxidation inNocardioides sp. Strain CF8

2001 ◽  
Vol 67 (11) ◽  
pp. 4992-4998 ◽  
Author(s):  
Natsuko Hamamura ◽  
Chris M. Yeager ◽  
Daniel J. Arp
Keyword(s):  
Northern Blot Analysis ◽  
Pcr Amplification ◽  
Alkane Oxidation ◽  
Pseudomonas Oleovorans ◽  
Alkane Hydroxylase ◽  
Degenerate Primers ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Degradation Activity ◽  
Binuclear Iron

ABSTRACT Alkane monooxygenases in Nocardioides sp. strain CF8 were examined at the physiological and genetic levels. Strain CF8 can utilize alkanes ranging in chain length from C2 to C16. Butane degradation by butane-grown cells was strongly inhibited by allylthiourea, a copper-selective chelator, while hexane-, octane-, and decane-grown cells showed detectable butane degradation activity in the presence of allylthiourea. Growth on butane and hexane was strongly inhibited by 1-hexyne, while 1-hexyne did not affect growth on octane or decane. A specific 30-kDa acetylene-binding polypeptide was observed for butane-, hexane-, octane-, and decane-grown cells but was absent from cells grown with octane or decane in the presence of 1-hexyne. These results suggest the presence of two monooxygenases in strain CF8. Degenerate primers designed for PCR amplification of genes related to the binuclear-iron-containing alkane hydroxylase fromPseudomonas oleovorans were used to clone a related gene from strain CF8. Reverse transcription-PCR and Northern blot analysis showed that this gene encoding a binuclear-iron-containing alkane hydroxylase was expressed in cells grown on alkanes above C6. These results indicate the presence of two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8.

scholarly journals Aplikasi Teknik Polymerase Chain Reaction (PCR) Menggunakan Primer Degenerate dan Spesifik Gen AV1 Untuk Mendeteksi Begomovirus Pada Tomat (Lycopersicon esculentum Mill.)

2015 ◽  
Vol 4 (3) ◽  
pp. 140 ◽  
Author(s):  
Tri J. Santoso ◽  
Sri H. Hidayat ◽  
M. Herman ◽  
, Sudarsono
Keyword(s):  
Lycopersicon Esculentum ◽  
Virus Transmission ◽  
Pcr Amplification ◽  
Degenerate Primer ◽  
Degenerate Primers ◽  
Specific Primers ◽  
West Java ◽  
Tomato Production ◽  
Central Java ◽  
Production Areas

<p>ABSTRACT</p><p>Begomoviruses infection has been reported and found in several important vegetable crops included  tomato.  Nevertheless,  the  information  of  detection and  identification  of  Begomovirus infecting  tomato  plants  in  some  of tomato  production  areas  using  PCR  technique  has  not  been widely reported.  The  objective  of  this  research  was  to  detect  Begomovirus infecting  tomatoes  in some of tomato production areas of East Java, Central Java, Special Province of Jogjakarta and West  Java  using  PCR  technique with degenerate  and  specific  primers.  PCR  amplification  of Begomovirus genome was conducted by using a pair of degenerate primers, i.e. PAL1v1978-F and PAR1c715-R  and  specific  primer,  AV1-F/R.  For  confirmation  the  virus, it  was  conducted  a  virus transmission from the symptomed plant tomato sampel to healthy plants by using whiteflies vector. The  results  of  this research  showed  that  the  symptomed  plants  collected  from  several tomato production  areas  of  East  Java,  Central  Java,  Special  Province  of Jogjakarta  and  West  Java indicated that those plants have been infected by Begomovirus following PCR detection using a pair of degenerate primers. The Begomovirus infection was indicated by the PCR amplified product with the size of 1500 bp.  The results of PCR amplification  using specific primers AV1-F/R to detect the genus  of  Begomovirus  showed  that  all  samples  of  plant collections  generated  of  780  bp  DNA fragment. Confirmation of the virus through transmission by  whitefiles  vectors in greenhouse from the symptomed plants and the positive PCR samples showed that the virus transmission process was succesfully conducted with indication of the emergence of symptoms in healthy plants.</p><p>Keywords: tomato  ( Lycopersicon  esculentum  Mill.) ,  Begomovirus, PCR technique, degenerate primer</p><p>ABSTRAK</p><p>Hasil  penelitian  sebelumnya  menunjukkan  bahwa  Infeksi  virus begomovirus di  beberapa tanaman sayuran termasuk tanaman tomat. Namun demikian, penelitian yang memberikan informasi mengenai  deteksi  dan  infeksi  dari virus  begomovirus  yang  menginfeksi  tanaman  tomat  dengan menggunakan teknik  PCR  masih  perlu  diteliti  lebih  lanjut.  Tujuan  dari  penelitian  adalah untuk mendeteksi begomovirus yang menginfeksi  tanaman  tomat di beberapa daerah produksi tomat dari Jawa Timur, Jawa Tengah,  Yogyakarta,  dan Jawa Barat menggunakan teknik PCR dengan primer degenerate  dan  spesifik. Amplifikasi  PCR  genom  begomovirus  dilakukan  dengan menggunakan sepasang  degenerate  primer  yaitu  PAL1v1978-F  dan   PAR1c715-R,  serta  primer  spesifik  adalah AV1-F/R.   Konfirmasi  virus dilakukan  dengan  teknik  penularan  virus  oleh  vektor  kutu  kebul ke tanaman sehat. Hasil penelitian menunjukkan bahwa  sampel-sampel tanaman tomat bergejala yang dikoleksi  dari  beberapa  daerah  di  Jawa Timur,  Jawa  Tengah,  Jawa  Barat  dan  D. I . Jogjakarta mengindikasikan adanya infeksi oleh Begomovirus setelah dideteksi menggunakan teknik PCR dengan primer degenerate.  Hasil amplifikasi PCR menggunakan primer spesifik AV1-F/R untuk mendeteksi  genus Begomovirus  menunjukkan  bahwa  semua sampel  tanaman  koleksi  menghasilkan  amplikon  yang berukuran  780  bp. Konfirmasi  virus  melalui  penularan  dengan  vektor  kutu  kebul  di  rumah kaca  dari sampel  tanaman  sakit  dan  positif  PCR  menunjukkan  terjadinya proses  penularan  virus  yang  ditandaidengan munculnya gejala-gejala pada tanaman yang sehat.</p><p>Kata kunci: Begomovirus, Lycopersicon esculentum, Primer degenerate, Tehnik PCR.</p>

scholarly journals Phylogenetic relationships among rhabdoviruses inferred using the L polymerase gene

2005 ◽  
Vol 86 (10) ◽  
pp. 2849-2858 ◽  
Author(s):  
H. Bourhy ◽  
J. A. Cowley ◽  
F. Larrous ◽  
E. C. Holmes ◽  
P. J. Walker
Keyword(s):  
Phylogenetic Analysis ◽  
Phylogenetic Relationships ◽  
Pcr Amplification ◽  
Polymerase Gene ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
The Family ◽  
Molecular Phylogenetic ◽  
History Of ◽  
Vector Borne

RNA viruses of the family Rhabdoviridae include arthropod-borne agents that infect plants, fish and mammals, and also include a variety of non-vector-borne mammalian viruses. Herein is presented a molecular phylogenetic analysis, the largest undertaken to date, of 56 rhabdoviruses, including 20 viruses which are currently unassigned or assigned as tentative species within the Rhabdoviridae. Degenerate primers targeting a region of block III of the L polymerase gene were defined and used for RT-PCR amplification and sequencing. A maximum-likelihood phylogenetic analysis of a 158-residue L polymerase amino acid sequence produced an evolutionary tree containing the six recognized genera of the Rhabdoviridae and also enabled us to identify four more monophyletic groups of currently unclassified rhabdoviruses that we refer to as the ‘Hart Park’, ‘Almpiwar’, ‘Le Dantec’ and ‘Tibrogargan’ groups. The broad phylogenetic relationships among these groups and genera also indicate that the evolutionary history of rhabdoviruses was strongly influenced by mode of transmission, host species (plant, fish or mammal) and vector (orthopteran, homopteran or dipteran).

Isolation and characterization of chalcone synthase (CHS) gene from Phalaenopsis and Doritaenopsis orchids

2020 ◽  
Vol 21 (11) ◽  
Author(s):  
Dewi Sukma ◽  
Aline Sisi Handini ◽  
Sudarsono Sudarsono
Keyword(s):  
Nucleotide Sequence ◽  
Chalcone Synthase ◽  
Pcr Amplification ◽  
Nucleotide Sequence Analysis ◽  
Degenerate Primers ◽  
Multiple Sequence ◽  
Isolation And Characterization ◽  
Key Enzyme ◽  
Young Leaves

Abstract. Sukma D, Handini AS, Sudarsono. 2020. Isolation and characterization of chalcone synthase (CHS) gene from Phalaenopsis and Doritaenopsis orchids. Biodiversitas 21: 5054-5064. Chalcone synthase (CHS) is a key enzyme in flavonoid biosynthesis. The research aims to isolate and characterize the CHS gene nucleotide sequence diversity of nine Phalaenopsis and one Doritaenopsis genotypes. Genomic DNA was isolated from young leaves and used to PCR amplify the gene using CHS specific degenerate primers. Results of PCR amplification yielded DNA fragments of 700 bp. Upon sequencing and nucleotide sequence analysis, we found that the genomic fragments were partial CHS gene from Phalaenopsis and Doritaenopsis genotypes and deposited the sequences in the NCBI Genebank Database accession numbers of KR184089-KR184098. More analysis of the sequences confirmed they shared ranged from 81-99% sequence identities to known CHS genes from Phalaenopsis deposited in the NCBI Database. They also shared 84-100% amino acid residues identity to CHS polypeptides. Multiple sequence alignment (MSA) and phylogenetic analysis further revealed the determined CHS nucleotide sequences from Phalaenopsis and Doritaenopsis genotypes were closely related to CHS genes from other orchid species.

Defeating numts: Semi-pure mitochondrial DNA from eggs and simple purification methods for field-collected wildlife tissues

Genome ◽  
2006 ◽  
Vol 49 (11) ◽  
pp. 1438-1450 ◽  
Author(s):  
Gabriela Ibarguchi ◽  
Vicki L. Friesen ◽  
Stephen C. Lougheed
Keyword(s):  
Mitochondrial Dna ◽  
Gradient Methods ◽  
Pcr Amplification ◽  
Cesium Chloride ◽  
Pcr Cloning ◽  
Degenerate Primers ◽  
Purification Methods ◽  
Mitochondrial Origin ◽  
Reference Sequences ◽  
Species Specific

Mitochondrial DNA (mtDNA) continues to play a pivotal role in phylogeographic, phylogenetic, and population genetic studies. PCR amplification with mitochondrial primers often yields ambiguous sequences, in part because of the coamplification of nuclear copies of mitochondrial genes (numts) and true mitochondrial heteroplasmy arising from mutations, hybridization with paternal leakage, gene duplications, and recombination. Failing to detect numts or to distinguish the origin of such homologous sequences results in the incorrect interpretation of data. However, few studies obtain purified mtDNA to confirm the mitochondrial origin of the first reference sequences for a species. Here, we demonstrate the importance and ease of obtaining semi-pure mtDNA from wildlife tissues, preserved under various typical field conditions, and investigate the success of 3 commercial extraction kits, cesium-chloride gradient mtDNA purification, long-template PCR amplification, cloning, and more species-specific degenerate primers. Using more detailed avian examples, we illustrate that unfertilized or undeveloped eggs provide the purest sources of mtDNA; that kits provide an alternative to cesium-chloride gradient methods; and that long-template PCR, cloning, and degenerate primers cannot be used to produce reliable mitochondrial reference sequences, but can be powerful tools when used in conjunction with purified mtDNA stocks to distinguish numts from true heteroplasmy.

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