• Title/Summary/Keyword: Fragment method

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Investigation of PCR-RFLPs within Major Histocompatibility Complex B-G Genes Using Two Restriction Enzymes in Eight Breeds of Chinese Indigenous Chickens

  • Xu, R.F.;Li, K.;Chen, G.H.;Qiang, B.Y.Z.;Mo, D.L.;Fan, B.;Li, C.C.;Yu, M.;Zhu, M.J.;Xiong, T.A.;Liu, Bang
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.7
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    • pp.942-948
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    • 2005
  • New polymorphism of major histocompatibility complex B-G genes was investigated by amplification and digestion of a 401bp fragment including intron 1 and exon 2 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique with two restriction enzymes of Msp I and Tas I in eight breeds of Chinese indigenous chickens and one exotic breed. In the fragment region of the gene, three novel single nucleotide polymorphisms (SNPs) were detected at the two restriction sites. We found the transition of two nucleotides of A294G and T295C occurred at Tas I restriction site, and consequently led to a non-synonymous substitution of asparagine into serine at position 54 within the deduced amino acid sequence of immunoglobulin variable-region-like domain encoded by the exon 2 of B-G gene. It was observed at rare frequency that a single mutation of A294G occurring at the site, also caused an identical substitution of amino acid, asparagine 54-to-serine, to that we described previously. And the transversion of G319C at Msp I site led to a non-synonymous substitution, glutamine 62-to-histidine. The new alleles and allele frequencies identified by the PCR-RFLP method with the two enzymes were characterized, of which the allele A and B frequencies at Msp I and Tas I loci were given disequilibrium distribution either in the eight Chinese local breeds or in the exotic breed. By comparison, allele A at Msp I locus tended to be dominant, while, the allele B at Tas I locus tended to be dominant in all of the breeds analyzed. In Tibetan chickens, the preliminary association analysis revealed that no significant difference was observed between the different genotypes identified at the Msp I and Tas I loci and the laying performance traits, respectively.

Characterization of Bacterial Community in the Ecosystem Amended with Phenol (페놀이 첨가된 생태계에서 세균 군집구조 변화의 분석)

  • 김진복;김치경;안태석;송홍규;이동훈
    • Korean Journal of Microbiology
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    • v.37 no.1
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    • pp.72-79
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    • 2001
  • The effect of phenol on the change of bacterial community in the effluent water from a wastewater treatment plant was analyzed by PCR and terminal restriction fragment length polymorphism (T-RFLP). The fragments of 16S rDNA were amplified by PCR with bacterial primers, where one of the primers was biotinylated at the 5'-end. After digestion with restriction enzymes, HaeIII and AluI, the biotinylated terminal restriction tragments (T-RFs) of the digested products were selectively isolated by using streptavidin paramagnetic particles. The single-stranded DNA of T-RFs was separated by electrophoresis on a polyacrylamide gel and detected by silver staining technique. When 10 standard strains were analyzed by our method, each strain had a unique T-RF which corresponded to the calculated size from the known sequences of RDP database. The T-RFLP fingerprint generated from the effluent water was very complex, and the predominant T-RFs corresponded to members of the genus Acinetobacter, Bacillus and Pseudomonas. In addition, the perturbation of bacterial community was observed when phenol was added to the sample at the final concentration of 250 $l^{-1}$. The number of T-RFs increased and the major bacterial population could be assigned to the genus Acinetobacter, Comamonas, Cytophaga and Pseudomonas. A intense band assigned to the putative genera of Acinetobacter and Cytophaga was eluted, amplified, and sequenced. The nucleotide sequence of the T-RF showed close relationship with the sequence of Acinetobacter junii.

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THE PROGNOSIS OF INTENTIONALLY RETAINED ROOT FRAGMENT OF PRIMARY TEETH (의도적으로 남겨진 유치근 파절편의 예후)

  • Lee, Suk-Hee;Kim, Shin;Jeong, Tae-Sung
    • Journal of the korean academy of Pediatric Dentistry
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    • v.34 no.1
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    • pp.156-161
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    • 2007
  • Pediatric dentists are frequently encountered with fractured root fragments of primary teeth caused either by the traumatic injuries or by the accidental fractures during the procedure of tooth extractions. In these situations, we often hesitate which method to choose, extract or retain it. In general, it is recommended to retain apical fragments, as the attempts to extract the apical fragments might harm the developing permanent tooth germ. This study was designed to ensure the validity of intentional retention of the root fragments of primary teeth in the situations described above. 6 children with intentionally root fragments who experienced root fracture in primary anterior teeth were available Periodic radiographic assessment was performed at 3 months interval for $7{\sim}37$ months. The results of this study showed that apical fragments had been resorbed through physiologic process in 5 patients. Apical fragment had been gingival emergence along with the erupting permanent tooth in 1 patient. There were no evidence of interference with eruption of permanent successors. In summary we have been ensured the validity of intentionally retention of the root fragments of primary teeth. Children with being remained apical root fragment should be recalled regularly for assessment and parents should be thoroughly informed about the situation with special emphasis on the necessity of periodic check-up.

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Transformation of Plant Cells by Gene Transfer : Construction of a Chimeric Gene Containing Deleted Maize Alcohol Dehydrogenase Intron and ${\beta}-Glucuronidase$ Gene and Its Expression in Potato (유전자 도입에 의한 식물세포의 형질전환 : 옥수수 알코올 탈수소효소 유전자의 절단된 인트론 및 ${\beta}-Glucuronidase$ 유전자를 함유하는 키메라 유전자의 제조와 감자에서의 발현)

  • 이광웅
    • Journal of Plant Biology
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    • v.35 no.3
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    • pp.237-245
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    • 1992
  • To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter and the effect of the deleted maize alcohol dehydrogenase I-S (Adhl-S) intron 1 on the expression of the CaMV $35S{\beta}-glucuronidase$ (GUS) gene in potato (Solanum tuberosum L. cv. Superior), we constructed a chimeric gene and transferred it into potato with Agrobacterium tumefaciens mediated method. The pLS201, a gene transfer vector of 17.7 kilobase pairs, was composed of the CaMV 35S promoter, the 249 base pairs of deleted maize Adhl-S intron 1, the GUS reporter gene, and the kanamycin resistance gene as a selectable marker for transformation. The GUS activity was examined by histochemical and spectrophotometric assay in transformed potato plants. The GUS activity was found primarily around the vascular tissue cells in stem and root. In the spectorophotometric assay, the level of GUS activity of transgenic potato transformed with CaMV 35S/249 bp of intron 1 fragment-GUS (pLS201) was compared with that of potato transformed with CaMV 35S-GUS (pBI121). The quantitative spectrophotometric assay showed that the level of GUS activity in potato transformed with pLS201 was higher in leaf, stem and root by 30-, 34- and 42-fold, respectively than those in potato transformed with pBI121. This results indicate that the inclusion of the deleted maize Adhl-S intron 1 resulted in increament of the GUS gene expression in transgenic potato.potato.

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Development of SCAR Marker for Discriminating between Violet Flowered Lines and White Flowered Lines in Chinese Bellflower (Platycodon grandiflorum A.) (청도라지와 백도라지의 구분을 위한 SCAR 마커 개발)

  • Park, Chun-Geon;Bang, Kyong-Hwan;Kim, Ok-Tae;Jin, Dong-Chun;Kim, Dong-Hwi;Sung, Jung-Sook;Seong, Nak-Sul;Park, Hee-Woon;Lee, Sang-Chul
    • Korean Journal of Medicinal Crop Science
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    • v.15 no.1
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    • pp.1-5
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    • 2007
  • To develop a convenient method for discriminating between violet flowered lines and white flowered lines in Chinese bellflower, RAPD analysis was carried out and SCAR markers were generated. Eighteen specific RAPD bands were obtained from 6 OPERON primer sets. Two out of eighteen RAPD bands were cloned into pGEM-T-Easy vectors and then subjected to the nucleotide sequence analysis. PgR1 and PgR2 DNA fragment, each specific for violet and white flowered lines, consist of 887 bp and 863 bp sequences, respectively. Two SCAR markers were developed from RAPD clones: SPgR1 (355 bp) from PgR1 and SPgR2 (493 bp) from PgR2. One (SPgR2) of these two markers was useful to differentiate between violet flowered lines and white flowered lines in Chinese bellflower.

Identification of Vegetable Oil-added Sesame Oil by a Mass Spectrometer-based Electronic Nose (Mass Spectrometer를 바탕으로 한 전자코를 이용한 식물성 유지가 혼합된 참기름의 판별 분석)

  • Son, Hee-Jin;Hong, Eun-Jeung;Ko, Sanghoon;Choi, Jin Young;Noh, Bong-Soo
    • Food Engineering Progress
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    • v.13 no.4
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    • pp.275-281
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    • 2009
  • Sesame oils are partially mixed with other vegetable oils due to high price in a Korean market. To find out authentic sesame oil, a mass spectrometer-based electronic nose (MS-based E-nose) was used. Sesame oil (Se) was blended with soybean oil (So) or corn oil (Co) at the ratio (Se:So, Se:Co) of 97:3, 94:6, 91:9, 88:12 and 85:15, respectively. Intensities of each fragment from sesame oil by MS-based E-nose were completely different from those of soybean oil or corn oil. The obtained results were used for discriminant function analysis (DFA). Volatile organic components (VOC) of soybean oil or corn oil were similar to those of fresh air and DFA plot indicated a significant separation of pure sesame oil and pure other oil. The group of the mixed oil was seperated with that of sesame oil in DFA plot and the added amount of soybean oil to sesame oil was correlated with discriminant function first score (DF1). MS based E-nose system could be used as an efficient method to investigate the purity of sesame oil.

Rapid detection of salmonellosis on serovar type of piglet with the polymerase chain reaction (중합효소연쇄반응을 이용한 자돈 혈청형에 따른 Salmonellosis의 신속한 검출)

  • Choi, Kyoung-seong;Park, Jin-ho;Kwon, Oh-deog;Lee, Joo-mook
    • Korean Journal of Veterinary Research
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    • v.38 no.4
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    • pp.763-770
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    • 1998
  • Salmonella typhimurium is a causitive agent of diarrhea, fever, gastroenteritis, septicemia and sudden death in piglet. The currently used methods such as IFA, ELISA, DNA hybridization assay is needed a long-time and difficult to detect the organism in carrier animal or contaminated sample with other agents. However, it is important to detect rapidly and sensitively S typhimurium in piglet with other infectious pathogens to minimize an economic loss. Two sets of PCR primer, rfbJ forward primer(5'-AGAATATGTAATTGTCAG-3') and reverse primer(5'-TAACCGTTTCAGTAGTTC-3') were designed to amplify a 882 by fragment of Salmonella serovar type B gene. The target genomic DNA for PCR was extracted from the cultivated materials with various enrichment periods in a nonselective enrichment agar and broth with clinical specimens. The PCR is carried out here made it possible to detect the gene from two hours. Also, the amplified fragment with PCR was cloned into pGEM-T vector and digested with restrict enzyme, and sequenced for the identification of Salmonella serotype B rfbJ gene. Duplicated cultivation agar-broth followed by PCR were performed to develop a rapid and sensitive detection of S typhlmurium based on serovar type. This duplicated cultivation-PCR method provides a sensitive and rapid diagnostic tool to detect Salmonella from infected piglet with improved sensitivity.

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Investigation of the Protonated State of HIV-1 Protease Active Site

  • Nam, Ky-Youb;Chang, Byung-Ha;Han, Cheol-Kyu;Ahn, Soon-Kil;No, Kyoung-Tai
    • Bulletin of the Korean Chemical Society
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    • v.24 no.6
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    • pp.817-823
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    • 2003
  • We have performed ab initio calculation on the active site of HIV-1 protease. The FEP method was used to determine the binding free energy of four different of protonated states of HIV-1 protease with inhibitor. The structure of the active site and hole structure was taken from the X-ray crystallographic coordinates of the C₂ symmetric inhibitor A74704 protease bound. The active site was modeled with the fragment molecules of binding pocket, acetic acid/ acetate anion (Asp25, Asp125), formamide (amide bond of Thr26/Gly27, Thr126/ Gly127), and methanol as inhibitor fragment. All possibly protonated states of the active site were considered, which were diprotonated state (0, 0), monoprotonated (-1, 0),(0, -1) and diunprotonated state (-1, -1). Once the binding energy Debind, of each model was calculated, more probabilistic protonated states can be proposed from binding energy. From ab-initio results, the FEP simulations were performed for the three following mutations: Ⅰ) Asp25 … Asp125 → AspH25 … Asp125, ⅱ) Asp25 … Asp125 → Asp25 … AspH125, ⅲ) AspH25 … Asp125 → AspH25 … AspH125. The free energy difference between the four states gives the information of the more realistic protonated state of active site aspartic acid. These results provide a theoretical prediction of the protonation state of the catalytic aspartic residues for A74707 complex, and may be useful for the evaluation of potential therapeutic targets.

Specific Marker Gene Analyses for DNA Polymorphism of the Blood Cell in Korea Native Brindled Cattle (칡한우 혈액에서 DNA 다양성 분석을 통한 표지 유전자 탐색)

  • Kim, Sang-Hwan;Hong, Yeon-Sik;Lee, Ho-Joun;Yoon, Jong-Taek
    • Development and Reproduction
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    • v.15 no.4
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    • pp.315-324
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    • 2011
  • This study was conducted to detect the specific expressing genes by using RAPD-PCR and RFLP method in the Korea Native Brindled Cattle, Korean Native cow and Holstein cattle. And then, the specific marker gene was investigated by the analysis of the genes for detection significance according to the expressing pattern. We found the specific expression gene by the RAPD-PCR analysis in Korea Native Brindled Cattle. It was detected the differences of the species in the colour and external section. The Korea Native Brindled Cattle were vary low compare to the Korean Native cow and Holstein cattle by analysis result of polymorphism and distribution. And there were a found the specific marker gene by sequencing in the R9B gene fragment of Korea Native Brindled Cattle. And the sequencing result of the R9B was different between Korean Native cow and Holstein cattle. Thus, this gene can be apply as the specific marker gene in the Korea Native Brindled Cattle.

The Treatment of One-Part Fractures of the Greater Thberosity of the Proximal Humerus (상완골 대결절 일분 골절의 치료)

  • Park Tae-Soo;Kim Tae-Seung;Park Ye-Soo;Kim Do-Hyeung;Kang Chang-Nam;Whang Kuhn-Sung
    • Clinics in Shoulder and Elbow
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    • v.2 no.1
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    • pp.21-27
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    • 1999
  • Purpose : The purpose of this study was to evaluate the functional outcomes of one-part fracture of the greater tuberosity that had been treated either by a conservative treatment or an operative approach. Materials and Method: Eighteen shoulders in 18 patients who had an one-part fracture of the greater tuberosity of the proximal humerus were managed, and the average follow-up period was 4 years and 10 months (range, 1 year to 8 years 6 months). Results: According to Neer's criteria for evaluation of results, in the group of 13 patients managed nonoperatively, the results were good or excellent in ten patients, fair in one, and poor in two. In the group managed operatively, the results were excellent in all five patients. Conclusion: If the displacement of the fragment is more than 5mm in young active patients, and more than 3mm especially in athletes and heavy laborers involved in overhead activity, the fragment should be mobilized, repaired and fixed into its original bed or a little bit inferolaterally with multiple heavy non-absorbable sutures, tension band technique, or cancellous screws and washers. We would suggest that the patients showing one-part fracture of the greater tuberosity of the proximal humerus should be evaluated individually.

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