• Title/Summary/Keyword: Primer extension

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PCR Primer Developed for Diagnosis of Xanthomonas arboricola pv. pruni in Prune (자두 검은점무늬병원균의 PCR진단 및 검출)

  • Ryu, Young-Hyun;Lee, Joong-Hwan;Kwon, Tae-Young;Kim, Seung-Han;Kim, Dong-Geun
    • Research in Plant Disease
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    • v.16 no.2
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    • pp.125-128
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    • 2010
  • Bacterial black spot disease of prune fruit (Prunus salicina cv. formosa) has outbroke around major prune production area, Gimcheon, Euiseong and Gunwi in Gyungbuk province and has caused severe economic loss. Integrons PCR primer was designed along with sample pre-incubation and nested PCR method to enhance detection sensitivity for early detection of bacteria in fields. Designed integrons PCR primer successfully detected Xanthomonas arboricola pv. pruni from field collected samples, fruit, leaf, branch and even in raindrop collected from prune orchard. Pre-incubation along with nested PCR enhanced sensitivity to detect X. arboricola pv. pruni from seemingly healthy looking, symptomless branches. Designed integrons PCR can be used in prune nursery fields and in plant quarantine practice for the detection of X. arboricola pv. pruni.

Single-base Discrimination Mediated by Proofreading Inert Allele Specific Primers

  • Lin-Ling, Chen;Zhang, Jia;Sommer, Steve S.;Li, Kai
    • BMB Reports
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    • v.38 no.1
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    • pp.24-27
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    • 2005
  • The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by $exo^+$ DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.

Multiplex Simple Sequence Repeat (SSR) Markers Discriminating Pleurotus eryngii Cultivar (큰느타리(Pleurotus eryngii) 품종 판별을 위한 초위성체 유래 다중 표지 개발)

  • Im, Chak Han;Kim, Kyung-Hee;Je, Hee Jeong;Ali, Asjad;Kim, Min-Keun;Joung, Wan-Kyu;Lee, Sang Dae;Shin, HyunYeol;Ryu, Jae-San
    • The Korean Journal of Mycology
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    • v.42 no.2
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    • pp.159-164
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    • 2014
  • For development of a method for differentiation of Pleurotus eryngii cultivars, simple sequence repeats (SSR) from whole genomic DNA sequence analysis was used for genotyping and two multiplex-SSR primer sets were developed. These SSR primer sets were employed to distinguish 12 cultivars and strains. Five polymorphic markers were selected based on the genotyping results. PCR using each primer produced one to four distinct bands ranging in size from 200 to 300 bp. Polymorphism information content (PIC) values of the five markers were in the range of 0.6627 to 0.6848 with an average of 0.6775. Unweighted pairgroup method with arithmetic mean clustering analysis based on genetic distances using five SSR markers classified 12 cultivars into two clusters. Cluster I and II were comprised of four and eight cultivars, respectively. Two multiplex sets, Multi-1 (SSR312 and SSR366) and Multi-2 (SSR178 and SSR277) completely discriminated 12 cultivars and strains with 21 alleles and a PIC value of 0.9090. These results might be useful in providing an efficient method for the identification of P. eryngii cultivars with separate PCR reactions.

Analysis of Transcripts Expressed from the UL47 Gene of Human Cytomegalovirus

  • Hyun, Jong-Jun;Park, Hyo-Soon;Kim, Ki-Ho;Kim, Hung-Jin
    • Archives of Pharmacal Research
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    • v.22 no.6
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    • pp.542-548
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    • 1999
  • The UL47 gene (b 60390-b 60388) located in the unique long region of the human cytomegalovirus (HCMV) AD169 strain genome was analyzed RNA mapping. Northern blot analysis showed that the UL47 gene was expressed at late times after infection (72 h postinfection). The 9.7-kb transcript was expressed in the infected cells but not in phosphonoformate-treated cells at 72 hpi, indicating that the UL47 gene was only expressed at late times after infection. To map the 5'-end and 3'-end of UL47 transcripts, primer at late times after infection. To map the 5'-end and 3'-end of UL47 transcripts, primer extension and RNase protection analysis were performed. Primer extension analysis revealed that the transcription initiation site of UL47 was located in 27 bp downstream (b 60323) of the TATA box motif. The sizes of UL47 ORF (approximately 2.9-kb) and UL48 ORF (approximately 6.7-kb) deduced from computer sequence analysis suggest that the expressed 9.7-kb transcript of UL47 uses the 3'-end polyadenylation signal of Ul48. The result of RNase protection determined that the 3'-end of UL47 RNA utilized the 3'-end polyadenylation signal of UL48, which is located in HCMV genome b 70082.

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Intraspecific Genetic Relation of Wasabia japonica Matsum. Based on RAPD Analysis (RAPD를 이용한 고추냉이의 유연관계 분석)

  • Heo, Su-Jeong;Kwon, Soon-Bae;Byeon, Hak-Soo;Seo, Jeong-Sik;Yoo, Ki-Oug
    • Korean Journal of Medicinal Crop Science
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    • v.12 no.1
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    • pp.31-35
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    • 2004
  • The genetic variation and intraspecific relationships between 10 individuals of seven cultivars and one Ulleungdo native of Wasabia japonica were investigated using RAPD (Randomly Amplified Polymorphic DNA) analysis. The 21 primers out of 50 random primers were amplified for all tested plants. The 68 (47.2%) among 144 bands derived from 21 primers showed polymorphism, and 3.2 bands per primer were observed. Number of bands per primer was ranged from 2 to 13, and average numbers were 6.8. The phenograms for 11 analyzed individuals by RAPD markers were not matched well with those of the result by morphological characters since they were clustered monophyletic at the similarity coefficient value ranged from 0.81 to 0.96. The Ulleungdo native individual was clustered sister to Daruma, Simanesairal, Sawa, and Hujidaruma cultivars. The RAPD markers were not useful to evaluate the intraspecific variations in Wasabia japonica cultivars, therefore need to more specific molecular phylogenetic characters such as AFLP technology and gene sequence of nuclear and chloroplast DNA.

Characterization of hrp2 + Gene Related to SNF2 Family in Schizosaccharomyces pombe (Schizosaccharomyces pombe에서 SNF2에 속하는 hrp2+ 유전자의 특성 연구)

  • Park, In-Soon
    • Environmental Mutagens and Carcinogens
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    • v.22 no.3
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    • pp.137-141
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    • 2002
  • The SNF2/SW12 family comprises proteins from a variety of species with in vivo functions, such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various types of DNA repair. This study was shown the characterization of hrp2+ gene which was isolated by PCR amplification using the conserved domain of SNF2 motifs. Sequence analysis of hrp2+ gene showed striking evolutionary conservation among the SNF2 family of proteins. The transcript of hrp2+ gene was found to be a 4.7 kb as identified by Northern hybridization. In addition, to determine the transcription initiation site of hrp2+ gene, primer extension analysis was performed. This result showed the band of 64 bp. The transcriptional start point was mapped to a position of 47 base pair from the first ATG codon of translational initiation codon. In order to investigate the inducibility of hrp2+ gene, transcript levels were examined after treating the cells to various DNA damaging agents. The transcripts of hrp2+ were induced by UV-irradiation. But the transcripts were not induced by treatment of 0.25% Methylmethane sulfonate (MMS). These results implied that the effects of damaging agents are complex and different regulatory pathways exist for the induction of this gene.

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On-off Regulation of 3' Exonuclease Excision to DNA Polymerization by Exo+ Polymerase

  • Zhang, Jia;Li, Kai
    • BMB Reports
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    • v.36 no.6
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    • pp.525-528
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    • 2003
  • The role of 3' exonuclease excision in DNA polymerization was evaluated in primer extensions using 3' allele-specific primers that had exonuclease-digestible and exonuclease-resistant 3' termini. With exonuclease-digestible unmodified 3' mismatched primers, the exo+ polymerase yielded template-dependent products. Using exonuclease-resistant 3' mismatched primers, no primer-extended product resulted from exo+ polymerase. As a control, polymerase without proofreading activity yielded primer-dependent products from 3' mismatched primers. These data indicated that a successful removal of the mismatch is required for DNA polymerization from the 3' mismatched primers by exo+ polymerase. In addition to the well-known proofreading from this mismatch removal, the premature termination in DNA polymerization, due to the failure of the efficient removal of the mismatched nucleotides, worked as an off-switch in maintaining the high fidelity in DNA replication from exo+ polymerase.

Analysis of Genetic Relationships of Korean Native Polygonatum spp. (우리 나라 자생 둥굴레속 식물의 유연관계 분석)

  • Jang, Kye-Hyun;Song, Geun-Woo;Chung, Jong-Il;Kang, Jin-Ho
    • Korean Journal of Medicinal Crop Science
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    • v.12 no.3
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    • pp.214-218
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    • 2004
  • Germplasms having valuable characters are increasjngly important for modern breeding programs. This study was conducted to obtain the basic data for effective use of genetic resources of Polygonatum. The relationship of seven Polygonatum species collected widely in Korea was analyzed by RAPD markers. Total number of alleles amplified by nineteen random primers were 114 to 157, and variation in number of alleles was also diverse among seven species examined. The seven species were divided into two groups; one was of Polygonatum stenophyllum and Polygonatum humile. the other was of Polygonatum inflatum, Polygonatum lasianthum, Polygonatum odoratum var. pluriflorum (1), (2), and for variegatum.

Development of Cleaved Amplified Polymorphic Sequence (CAPS) Marker for Selecting Powdery Mildew-Resistance Line in Strawberry (Fragaria×ananassa Duchesne) (딸기 흰가루병 저항성 계통 선발을 위한 분자마커 개발)

  • Je, Hee-Jeong;Ahn, Jae-Wook;Yoon, Hae-Suk;Kim, Min-Keun;Ryu, Jae-San;Hong, Kwang-Pyo;Lee, Sang-Dae;Park, Young-Hoon
    • Horticultural Science & Technology
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    • v.33 no.5
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    • pp.722-729
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    • 2015
  • Powdery mildew (PM) caused by Podosphaera aphanis is a major disease that can result in significant yield losses in strawberry (Fragaria ${\times}$ ananassa Duchesne). For preventing PM, pesticides are usually applied in strawberry. In this study, molecular markers were developed to increase breeding efficiency of PM-resistance cultivars by marker-assisted selection (MAS). An $F_2$ population derived from a cross between PM-resistance 'Seolhyang' and PM-susceptibility 'Akihime' was evaluated for disease resistance to PM and RAPD (random amplification of polymorphic DNA)-BSA (bulked segregant analysis). Among 200 RAPD primers tested, OPE10 primer amplified a 311bp-band present in with 331bp. Sequence alignment performed for searching polymorphisms and six single nucleotide polymorphism (SNP) were found in amplified regions. To develop polymorphic marker for distinguishing between resistant and susceptible, RAPD was converted to cleaved amplified polymorphic sequence (CAPS) marker. Among restriction enzymes associated with six SNPs, Eae I (Y/GGCCR) was successfully digested to 231bp in susceptible. The results suggest that the selected CAPS marker could be used for increasing efficiency of selecting powdery mildew resistant strawberry in breeding system.