• 제목/요약/키워드: primer extension

검색결과 123건 처리시간 0.019초

흑한우와 한우 및 수입우를 판별하기 위한 multiplex PCR 기술 (PCR Technique for Determining Jeju Black Cattle, Hanwoo and Imported Beef)

  • 김찬수;고정문;차현철;박중국;정준
    • 생명과학회지
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    • 제24권8호
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    • pp.910-914
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    • 2014
  • 본 연구는 현행 한우확인시험법에 이용되는 Microsatellite (MS)와 소의 유전형질 중 품종 판별에 주로 이용되는 Melanocortin Receptor 1 (MC1R) 유전자상의 Single Nucleotide Polymorphism (SNP)를 분석하여 설계한 Primer를 Multiplex PCR을 활용하여 소의 품종을 판별하였다. MC1R 유전자형은 $E^D$, $E^+$, e형의 3Type으로 나뉘며 $E^D/E^D$, $E^D/E^+$, $E^D/e$, $E^+/E^+$, $E^+/e$, e/e의 6가지 유전자형을 가진다. $E^D$유전자형은 외래종이 지닌 유전자형으로 $E^D/E^D$, $E^D/E^+$, $E^D/e$이 이에 속하며, e유전자형은 한우가 지닌 유전자형으로 e/e의 유전자형을 가진다. 흑한우의 경우 $E^D$, $E^+$, e의 모든 유전자형을 가지고 있으나 이는 교잡에 의해 나타난 것으로 보이며 $E^+$유전자형이 흑한우 고유의 유전자형으로 추정되어 본 연구에서는 이에 따라 $E^+/E^+$, $E^+/e$의 유전자형을 흑한우로 분류하였다. 그러나 $E^D$, $E^+$의 경우 단순 PCR기법만으로는 그 판별에 어려움이 있어, MS Maker를 활용한 다형성 분석을 통해 흑한우와 수입우를 판별할 수 있는 새로운 Primer를 설계하였으며, 이를 통해 소의 품종을 판별하였다.

Optimization of SNP Genotyping Assay with Fluorescence Polarization Detection

  • Cai Chun Mei;Van Kyujung;Kim Moon Young;Lee Suk-Ha
    • 한국작물학회지
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    • 제50권5호
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    • pp.361-367
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    • 2005
  • Single nucleotide polymorphisms (SNPs) are valuable DNA markers due to their abundance and potential for use in automated high-throughput genotyping. Numerous SNP genotyping assays have been developed. In this report, one of effective and high throughput SNP genotyping assays, which was named the template-directed dye-terminator incorporation with fluorescence polarization detection (FP-TDI) was described. Although the most of this assay succeed, the objective of this work was to deter­mine the reasons for the failures, find ways to improve the assay and reduce the running cost. Ninety $F_2$-derived soybean, Glycine max (L.) Merr., RILs from a cross between 'Pureunkong' and 'Jinpumkong 2' were genotyped at four SNPs. FP measurement was done on $Victot^3$ microplate reader (perkinelmer Inc., Boston, MA, USA). Increasing the number of thermal cycles in the single-base extension step increased the separation of the FP values between the products corresponding to different genotypes. But in some assays, excess of heterozygous genotypes was observed with increase of PCR cycles. We discovered that the excess heterozygous was due to misincorporation of one of the dye­terminators during the primer extension reaction. After pyrophosphatase incubation and thermal cycle control, misincoporation can be effectively prevented. Using long amplicons instead of short amplicons for SNP genotyping and decreasing the amount of dye terminator and Acyclopol Taq polymerase to 1/2 or 1/3 decreased the cost of the assay. With these minor adjustments, the FP-TDI assay can be used more accurately and cost-effectively.

Rapid Identification of Ginseng Cultivars (Panax ginseng Meyer) Using Novel SNP-Based Probes

  • Jo, Ick-Hyun;Bang, Kyong-Hwan;Kim, Young-Chang;Lee, Jei-Wan;Seo, A-Yeon;Seong, Bong-Jae;Kim, Hyun-Ho;Kim, Dong-Hwi;Cha, Seon-Woo;Cho, Yong-Gu;Kim, Hong-Sig
    • Journal of Ginseng Research
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    • 제35권4호
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    • pp.504-513
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    • 2011
  • In order to develop a novel system for the discrimination of five ginseng cultivars (Panax ginseng Meyer), single nucleotide polymorphism (SNP) genotyping assays with real-time polymerase chain reaction were conducted. Nucleotide substitution in gDNA library clones of P. ginseng cv. Yunpoong was targeted for the SNP genotyping assay. From these SNP sites, a set of modified SNP specific fluorescence probes (PGP74, PGP110, and PGP130) and novel primer sets have been developed to distinguish among five ginseng cultivars. The combination of the SNP type of the five cultivars, Chungpoong, Yunpoong, Gopoong, Kumpoong, and Sunpoong, was identified as 'ATA', 'GCC', 'GTA', 'GCA', and 'ACC', respectively. This study represents the first report of the identification of ginseng cultivars by fluorescence probes. An SNP genotyping assay using fluorescence probes could prove useful for the identification of ginseng cultivars and ginseng seed management systems and guarantee the purity of ginseng seed.

Incidence of Viral Diseases and Occurrence of Three Unreported Viruses in Yams in Korea

  • Lee, Joong-Hwan;Park, Chung Youl;Cho, Ha-Jeong;Oh, Jonghee;Kim, Bong-Sub;Park, Eun Hey;Son, Chang-Gi;Lee, Su-Heon
    • 식물병연구
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    • 제23권1호
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    • pp.82-87
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    • 2017
  • During 2012 to 2014, a survey for the presence of viral diseases in yam plants was carried out in a field of the Institute for Bioresources Research in Gyeongsangbuk-do, Korea. A total of 88 leaf samples were collected and tested by reverse transcription polymerase chain reaction using specific primer sets. Eighty-one samples were positive for Broad bean wilt virus 2 (BBWV2), Chinese yam necrotic mosaic virus (ChYNMV), Cucumber mosaic virus (CMV), Japanese yam mosaic virus (JYMV), and Yam mild mosaic virus (YMMV), whereas Yam mosaic virus (YMV) was not detected. Additionally, seven samples were negative for all viruses. Several samples exhibited mixed (double and triple) infections. Three viruses (CMV, JYMV, and YMMV) were detected for the first time in yam plants in Korea. A BLAST search showed that three viruses shared nucleotide identities with CMV-Ca (98%), JYMV-O2 (91%), and YMMV-TG_NH_1 (86%). Thus, our findings confirmed that yam plants cultivated in Korea were infected with multiple viruses with three of these viruses reported for the first time in Korea.

Characterization of Melon necrotic spot virus Occurring on Watermelon in Korea

  • Kwak, Hae-Ryun;Kim, Jeong-Soo;Cho, Jeom-Deog;Lee, Joong-Hwan;Kim, Tae-sung;Kim, Mi-Kyeong;Choi, Hong-Soo
    • The Plant Pathology Journal
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    • 제31권4호
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    • pp.379-387
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    • 2015
  • Melon necrotic spot virus (MNSV) was recently identified on watermelon (Citrullus vulgaris) in Korea, displaying as large necrotic spots and vein necrosis on the leaves and stems. The average occurrence of MNSV on watermelon was found to be 30-65% in Hapcheon and Andong City, respectively. Four isolates of the virus (MNSV-HW, MNSV-AW, MNSV-YW, and MNSV-SW) obtained from watermelon plants in different areas were non-pathogenic on ten general indicator plants, including Chenopodium quinoa, while they infected systemically six varieties of Cucurbitaceae. The virus particles purified by 10-40% sucrose density gradient centrifugation had a typical ultraviolet spectrum, with a minimum at 245 nm and a maximum at 260 nm. The morphology of the virus was spherical with a diameter of 28-30 nm. Virus particles were observed scattered throughout the cytoplasm of watermelon cells, but no crystals were detected. An ELISA was conducted using antiserum against MNSV-HW; the optimum concentrations of IgG and conjugated IgG for the assay were $1{\mu}l/ml$ and a 1:8,000-1:10,000 dilutions, respectively. Antiserum against MNSV-HW could capture specifically both MNSV-MN from melon and MNSV-HW from watermelon by IC/RT-PCR, and they were effectively detected with the same specific primer to produce product of 1,172 bp. The dsRNA of MNSV-HW had the same profile (4.5, 1.8, and 1.6 kb) as that of MNSV-MN from melon. The nucleotide sequence of the coat protein of MNSV-HW gave a different phylogenetic tree, having 17.2% difference in nucleotide sequence compared with MNSV isolates from melon.

국내 승인 LM면화의 자연환경 모니터링을 위한 multiplex PCR 개발 (Multiplex PCR method for environmental monitoring of approved LM cotton events in Korea)

  • 조범호;설민아;신수영;김일룡;최원균;엄순재;송해룡;이중로
    • Journal of Plant Biotechnology
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    • 제43권1호
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    • pp.91-98
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    • 2016
  • 면화(cotton)는 섬유를 수확하고, 면실유는 식용으로 가공 후 남은 것은 다시 사료로 이용되어 다방면으로 다양하게 활용되는 작물이다. LM 면화는 옥수수, 대두, 캐놀라와 달리 아시아권(중국, 인도 등)에서 가장 많이 재배되고 있으며, 우리나라는 2013년까지 세계 1위의 LM 면실(사료용) 수입국이며, 세계 4위의 면실박 수입국으로 해마다 증가하는 LM 면화의 수입량과 맞물려 유통 및 소비과정에서의 비의도적으로 유출 가능성이 증가함에 따라 LM 면화의 자연생태계 위해성 평가 및 안전관리가 요구된다. 본 연구에서는 국내 수입 승인 LM 면화 6개 이벤트(MON15985, MON531, GHB614, LLCOTTON25, MON88913, MON1445)의 동시증폭 검출법(multiplex PCR)을 개발하여 보다 신속하고 명확한 검출기법을 확립하고자 하였다. 최적 multiplex PCR 반응 조건은 2개 LM 면화 이벤트 MON15985 (214 bp), MON531 (270 bp)와 4개 LM 면화 이벤트 GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), MON1445 (389 bp)가 한번의 반응에 명확하게 검출되는 최적 반응 조건 및 primer 반응 농도의 조절을 통해 서로 다른 생성물 크기로 명확히 구분되도록 하였고, 최적 primer 농도는 반응액 최종농도 0.2~0.66 pmol로 primer 쌍 마다 각각 다른 최적 농도 조합의 cocktail을 만들어 활용하였다. Duplex PCR 반응 조건은 초기 $95^{\circ}C$ 5분 반응 후, $95^{\circ}C$ 15초, $55^{\circ}C$ 20초 15회 반응하고, 다시 $95^{\circ}C$ 15초, $60^{\circ}C$ 20초 25회 반응하였을 때 최적 검출이 이루어졌고, tetraplex PCR 반응 조건은 $95^{\circ}C$ 5분 반응 후, $95^{\circ}C$ 15초, $60^{\circ}C$ 20초 50회 반응하였을 때 최적 검출이 이루어졌다. 본 연구에서 개발된 multiplex PCR 검출법은 국내 수입 유통 LM 면화의 자연환경 모니터링에 활용함에 있어 요구되는 연구인력, 시간 및 비용을 보다 효율적으로 개선하는데 적용될 수 있을 것으로 사료된다.

Isolation and Characterization of the sod2$^{2+}$ Gene Encoding a Putative Mitochondrial Manganese Superoxide Dismutase in Schizosaccharomyces bombe

  • Jeong, Jae-Hoon;Kwon, Eun-Soo;Roe, Jung-Hye
    • Journal of Microbiology
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    • 제39권1호
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    • pp.37-41
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    • 2001
  • The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

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sanN Encoding a Dehydrogenase is Essential for Nikkomycin Biosynthesis in Streptomyces ansochromogenes

  • Ling, Hong-Bo;Wang, Guo-Jun;Li, Jin-E;Tan, Hua-Rong
    • Journal of Microbiology and Biotechnology
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    • 제18권3호
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    • pp.397-403
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    • 2008
  • Nikkomycins are a group of peptidyl nucleoside antibiotics with potent fungicidal, insecticidal, and acaricidal activities. sanN was cloned from the partial genomic library of Streptomyces ansochromogenes 7100. Gene disruption and complementation analysis demonstrated that sanN is essential for nikkomycin biosynthesis in S. ansochromogenes. Primer extension assay indicated that sanN is transcribed from two promoters (sanN-P1 and sanN-P2), and sanN-P2 plays a more important role in nikkomycin biosynthesis. Purified recombinant SanN acts as a dehydrogenase to convert benzoate-CoA to benzaldehyde in a random-order mechanism in vitro, with respective $K_{cat}/K_m$$ values of $3.8mM^{-1}s^{-1}\;and\;12.0mM^{-1}s^{-1}$ toward benzoate-CoA and NADH, suggesting that SanN catalyzes the formation of picolinaldehyde during biosynthesis of nikkomycin X and Z components in the wild-type stain. These data would facilitate us to understand the biosynthetic pathway of nikkomycins and to consider the combinatorial synthesis of novel antibiotic derivatives.

Quantification of the galactose-operon mRNAs 5 bases different in their 5'-ends

  • Ji, Sang-Chun;Wang, Xun;Jeon, Heung-Jin;Yun, Sang-Hoon;Lee, Hee-Jung;Lim, Heon-M.
    • BMB Reports
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    • 제43권7호
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    • pp.474-479
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    • 2010
  • Three assay methods for quantification of the two galactose-operon mRNAs that only differ by 5 bases in their 5'-end are presented. The 5' ends of each mRNA were extended by ligating the 3'-end of the abundant 5S rRNA. This ligation extends the 5' ends of the two gal mRNAs long enough to be distinguished by the specific PCR primers in the following quantification reactions. Quantification of the corresponding cDNAs was performed either by primer extension assay or real-time qPCR. To circumvent the problem of the RNA ligation reaction (i.e. very low ligation efficiency), we devised a new method that employs real-time qPCR directly for the quantification of the gal transcripts which differ by 5 bases in their 5'-ends.

Cloning and Molecular Characterization of groESL Heat-Shock Operon in Methylotrophic Bacterium Methylovorus Sp. Strain SS1 DSM 11726

  • Eom, Chi-Yong;Kim, Eung-Bin;Ro, Young-Tae;Kim, Si-Wouk;Kim, Young-Min
    • BMB Reports
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    • 제38권6호
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    • pp.695-702
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    • 2005
  • The groESL bicistronic operon of a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 was cloned and characterized. It was found to consist of two ORFs encoding proteins with molecular masses of 11,395 and 57,396 daltons, which showed a high degree of homology to other bacterial GroES and GroEL proteins. The genes were clustered in the transcription order groES-groEL. Northern blot analyses suggested that the groESL operon is transcribed as a bicistronic 2.2-kb mRNA, the steady-state level of which was markedly increased by temperature elevation. Primer extension analysis demonstrated one potential transcription start site preceding the groESL operon, which is located 100bp upstream of the groES start codon. The transcription start site was preceded by a putative promoter region highly homologous to the consensus sequences of Escherichia coli ${\sigma}^{32}$-type heat shock promoter, which functioned under both normal and heat shock conditions in E. coli. Heat shock mRNA was maximally produced by Methylovorus sp. strain SS1 approximately 10min after increasing the temperature from 30 to $42^{\circ}C$. The groESL operon was also induced by hydrogen peroxide or salt shock.