• Title/Summary/Keyword: degenerate PCR primers

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Genetic variation of BIV isolates characterized by PCR using degenerate primers

  • Kwon, Oh-Sik;Sninsky, John J.
    • Journal of Microbiology
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    • v.33 no.3
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    • pp.252-259
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    • 1995
  • The PCR was employed to detect and characterize the bovine immunodeficiency-like virus (BIV), which is a newly recognized member of the I entivirinae of the retroviruses. Degenerate primers representing the conserved regions in the pol genes of the Lentivirinae, were used to detect proviral DNA obtained from the bovine embryonic spleen cell cultures infected with BIV. The PCR amplified DNA fragment was molecularly cloned and sequenced. The BIV DNA fragment contained a sequence identical to that reported by Garvey et al. (Garvey et al., 1990. Virology, 175, 391-409). With the degenerate primers, peripheral blood mononuclear cells (PBMCs) of sick cattle and cells cultured with BIV were tested to determine genetic variation of BIV pol conserved sequence. We found the sequence heterogeneity within cultures and most variations occurred at the third base of codons that would not lead to amino acid substitutions. Another change was GAG (Glu) to AAG (Lys) within the BIV isolates. Interestingly, the altered sequence is also found in other lentiviruses such as HIV-2, SIV mac, CAEV and EIAV.

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Rapid Detection and Isolation of Known and Putative $\alpha-L-Arabinofuranosidase$ Genes Using Degenerate PCR Primers

  • Park, Jung-Mi;Han, Nam-Soo;Kim, Tae-Jip
    • Journal of Microbiology and Biotechnology
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    • v.17 no.3
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    • pp.481-489
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    • 2007
  • [ $\alpha$ ]-L-Arabinofuranosidases (AFases; EC 3.2.1.55) are exo-type enzymes, which hydrolyze terminal nonreducing arabinose residues from various polysaccharides such as arabinan and arabinoxylan. Genome-wide BLAST search showed that various bacterial strains possess the putative AFase genes with well-conserved motif sequences at the nucleotide and amino acid sequence levels. In this study, two sets of degenerate PCR primers were designed and tested to detect putative AFase genes, based on their three highly conserved amino acid blocks (PGGNFV, GNEMDG; and DEWNVW). Among 20 Bacillus-associated species, 13 species were revealed to have putative AFase genes in their genome and they share over 67% of amino acid identities with each other. Based on the partial sequence obtained from an isolate, an AFase from Geobacillus sp. was cloned and expressed in E. coli. Enzymatic characterization has verified that the resulting enzyme corresponds to a typical AFase. Accordingly, degenerate PCR primers developed in this work can be used for fast, easy, and specific detection and isolation of putative AFase genes from bacterial cells.

Search for [NiFe]-Hydrogenase using Degenerate Polymerase Chain Reaction (Degenerate Polymerase Chain Reaction을 통한 [NiFe]-Hydrogenase의 탐색)

  • Jung, Hee-Jung;Kim, Jaoon Y.H.;Cha Hyung-Joon
    • 한국신재생에너지학회:학술대회논문집
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    • 2005.11a
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    • pp.631-633
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    • 2005
  • For biohydrogen production, hydrogenase is a key enzyme. In the present work we performed search of [NiFe]-hydrogenases from hydrogen producing microorganisms using degenerate polymerase chain reaction (PCR) strategy. Degenerate primers were designed from the conserved region of [NiFe]-hydrogenase group I especially on structural genes encoding for catalytic subunit of [NiFe]-hydrogenase from bacteria producing hydrogen. Most of [NiFe]-hydrogenase (group I) are expressed via complex mechanism with aid of auxiliary protein and localized through twin-arginine translocation pathway. [NiFe]-hydrogenase is composed of large and small subunits for catalytic activity. It is known that only small subunit has signal peptide for periplasmic localization and large & small subunitscome together before localization. During this process, large subunit is treated by endopeptidase for maturation. Based on these information we used signal peptide sequence and C-terminal of large subunit by recognized by endopeptidase as templates for degenerate primers. About 2,900 bp of PCR products were successfully amplified using the designed degenerate primers from genomic DNAs of several microorganisms. The amplified PCR products were inserted into T-vector and then sequenced to confirm.

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PCR Cloning of Genes Encoding the Mn-Peroxidase Isozyme Family from Trametes versicolor KN9522 Using Degenerate Primers (구름버섯균 KN9522에서 degenerate primer를 이용한 Mn-Peroxidase 동위효소 유전자들의 PCR 클로닝)

  • Jun, Sang-Cheol;Kim, Kyu-Joong
    • Korean Journal of Microbiology
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    • v.42 no.1
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    • pp.77-81
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    • 2006
  • Degenerate primers corresponding to the sequences of the N-terminal regions of Mn-peroxidase isozymes were used to isolate the genomic fragments encoding the isozymes of Mn-peroxidase, CVMP1, CVMP2, CVMP3 and CVMP5 from the white-rot fungus Trametes versicolor KN9522. Three isozymes except one gave the expected PCR products (cmp1, cmp2 and cmp5) of about 900 base pairs, respectively. DNA sequence data obtained from each PCR products were used to analyze the BLAST program search on the National Center for Biotechnology Information. cmp1, cmp2 and cmp5 were similar to MPG-I (GenBank accession number Z30668) and PGV-II (GenBank accession number, Z54279) gene T. versicolor PRL572. PCR products of cmp1 and cmp2 showed 77%, 95% base sequence similarities to MPG-I gene and cmp5 showed about 88% similarity to PGV-II gene from T. versicolor PRL572. From this experiment, we could isolate genomic DNA fragments with degenerate primers designed from the N-terminal amino acid sequences of Mn-peroxidase isozyme family.

Isolation of Cryptic Polyene Hydroxylase Gene in Rare Actinomycetes via Polyene-specific Degenerate PCR. (Polyene 특이적인 PCR에 의한 희소 방선균 유래 Cryptic Polyene Hydroxylase 유전자의 분리)

  • 박현주;명지선;박남실;한규범;김상년;김응수
    • Microbiology and Biotechnology Letters
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    • v.32 no.3
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    • pp.282-285
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    • 2004
  • The polyene antibiotics including nystatin, pimaricin, amphotericin and candicidin are a family of most promising antifungal polyketide compounds, typically produced by rare actinomycetes species. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly homologous biosynthetic genes among polyene-producers such as polyketide synthase (PKS) and cytochrome P450 hydroxylase (CYP) genes. Based on amino acid sequence alignment among actinomycetes CYP genes, the highly-conserved regions specific for only polyene CYP genes were identified and chosen for degenerate PCR primers, followed by the PCR-screening with various actinomycetes genomic DNAs. Among tested several polyene non-producing actinomycetes strains, Pseudonorcardia autotrophica strain was selected based on the presence of PCR product with polyene-specific CYP gene primers, and then confirmed to contain a cryptic novel polyene hydroxylase gene in the chromosome. These results suggest that the polyene-specific hydroxylase gene PCR should be an efficient way of screening and isolating potentially-valuable cryptic polyene antibiotic biosynthetic genes from various microorganisms including rare actinomycetes.

RFLP Analysis of cry1 and cry2 Genes of Bacillus thuringiensis Isolates from India

  • Patel, Ketan D.;Ingle, Sanjay S.
    • Journal of Microbiology and Biotechnology
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    • v.22 no.6
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    • pp.729-735
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    • 2012
  • The PCR-RFLP method has been useful for detection of known genes and identification of novel genes. In the present study, degenerate primers were designed from five groups of cry1 genes for PCR-RFLP analysis. Bacillus thuringiensis (Bt) isolates from different regions were evaluated for PCR amplification of various cry1 genes using newly designed primers and cry2 genes using reported primers. PCR analysis showed an abundance of cry1A genes and especially cry1Ac genes in isolates from all regions. RFLP analysis revealed the presence of multiple cry1A genes in isolates from central and southern regions. Unique digestion patterns of cry1A genes were observed in isolates from each region. Few of the isolates represented a digestion pattern of cry1A genes that did match to any of the known cry1A genes. RFLP analysis suggested an abundance of cry2Ab along with a novel cry2 gene in Bt isolates from different regions of India. Sequence analysis of the novel cry2 gene revealed 95% sequence identity to cry2Ab and cry2Ah genes. Phylogenetic analysis revealed that the novel cry2 gene could have diverged earlier than the other cry2 genes. Our results encourage finding of more diverse cry2 genes in Bt isolates. Rarefaction analysis was used to compare cry1A gene diversity in isolates from different soil types. It showed a higher degree of cry1A gene diversity in isolates from central region. In the present study, we propose the use of novel degenerate primers for cry1 genes and the PCR-RFLP method using a single enzyme to distinguish multiple cry1A and cry2 genes as well as identify novel genes.

Novel pan-lineage VP1 specific degenerate primers for precise genetic characterization of serotype O foot and mouth disease virus circulating in India

  • Sagar Ashok Khulape;Jitendra Kumar Biswal;Chandrakanta Jana;Saravanan Subramaniam;Rabindra Prasad Singh
    • Journal of Veterinary Science
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    • v.24 no.3
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    • pp.40.1-40.6
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    • 2023
  • Analysis of the VP1 gene sequence of the foot and mouth disease virus (FMDV) is critical to understanding viral evolution and disease epidemiology. A standard set of primers have been used for the detection and sequence analysis of the VP1 gene of FMDV directly from suspected clinical samples with limited success. The study validated VP1-specific degenerate primer-based reverse transcription polymerase chain reaction (RT-PCR) for the qualitative detection and sequencing of serotype O FMDV lineages circulating in India. The novel degenerate primer-based RT-PCR amplifying the VP1 gene can circumvent the genetic heterogeneity observed in viruses after cell culture adaptation and facilitate precise viral gene sequence analysis from clinical samples.

Development of Detection Method for Cyclomaltodextrinase Family Genes using Degenerate PCR Primers

  • Oh, Su-Won;Jang, Myoung-Uoon;Jeong, Chang-Ku;Yuk, Jeong-Bin;Park, Jung-Mi;Park, Kwan-Hwa;Kim, Tae-Jip
    • Food Science and Biotechnology
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    • v.15 no.6
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    • pp.967-974
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    • 2006
  • Cyclomaltodextrinases (CDases), maitogenic amylases, and neopullulanases share highly conserved primary structures and similar characteristics, and are thus classified into the same family. BLAST search has showed that a variety of bacterial strains harbor putative CDase family genes with several well-conserved motif amino acid sequences. In this study, four degenerate polymerase chain reaction (PCR) primer sets were designed for the detection of CDase genes, on the basis of their highly conserved amino acid blocks (WYQIFP, DGWRLD, LGSHDT, and KCMVW). The PCR detection conditions were optimized and the detection specificity of each for the primer sets was tested against the genomic DNAs isolated from 23 different Bacillus-associated species. Consequently, all tested primer sets evidenced successful amplification of specific PCR products in length, which share 55-98% amino acid sequence identity with known and putative CDases. The primers developed herein, therefore, can be applied for the easy and efficient detection and isolation of CDase family genes for the modification of functional food carbohydrates.

Convenient Virion Capture (VC)/PCR for Tomato yellow leaf curl geminivirus Occurring on Tomato in Korea (우리나라 토마토에 발생한 토마토황화잎말림바이러스(Tomato yellow leaf curl geminivirus)의 초간편 Virion Capture(VC)/PCR 진단법)

  • Cho, Jeom-Deog;Kim, Tae-Seong;Kim, Ju-Hee;Choi, Gug-Seoun;Chung, Bong-Nam;Choi, Hong-Soo;Kim, Jeong-Soo
    • Research in Plant Disease
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    • v.14 no.3
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    • pp.233-237
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    • 2008
  • Tomato yellow leaf curl virus (TYLCV), a newly reported Geminivirus from tomato, generated recently large economic losses in Korea. Development of a fast and precise genetic diagnosis technique for detecting TYLCV which Agricultural research and extension services can utilize easy and handy is very important to prevent yield losses. Virion Capture (VC)/PCR is a simple, accurate and economical genetic detection method without any works or commercial kits for the extraction of the nucleic acid from the infected plants. Primers of twenty two for detection of TYLCV were designed and tested with extracted total DNA or crude sap from tomato leaf infected with TYLCV and healthy plant. Nine primers for total DNA using conventional PCR and another 9 primers for VC/PCR were selected eventually. Primers of six having same specificity were selected from the two methods and tested with other Geminivirus, Tobacco leaf curl virus (TLCV) by VC/PCR. Finally specific primers of four were selected for detection of TYLCV using VC/PCR, and Deng (540, 541), a degenerate primer for Geminivirus reported in 1996, was also developed for VC/PCR.

Divergent long-terminal-repeat retrotransposon families in the genome of Paragonimus westermani

  • Bae, Young-An;Kong, Yoon
    • Parasites, Hosts and Diseases
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    • v.41 no.4
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    • pp.221-231
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    • 2003
  • To gain information on retrotransposons in the genome of Paragonimus westermani, PCR was carried out with degenerate primers, specific to protease and reverse transcriptase (rt) genes of long-terminal-repeat (LTR) retrotransposons. The PCR products were cloned and sequenced, after which 12 different retrotransposon-related sequences were isolated from the trematode genome. These showed various degrees of identity to the polyprotein of divergent retrotransposon families. A phylogenetic analysis demonstrated that these sequences could be classified into three different families of LTR retrotransposons, namely, Xena, Bel, and Gypsy families. Of these, two mRNA transcripts were detected by reverse transcriptase-PCR, showing that these two elements preserved their mobile activities. The genomic distributions of these two sequences were found to be highly repetitive. These results suggest that there are diverse retrotransposons including the ancient Xena family in the genome of P. westermani, which may have been involved in the evolution of the host genome.