• Title/Summary/Keyword: longer primer

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Prognostic Significance of GSTP1, XRCC1 and XRCC3 Polymorphisms in Non-small Cell Lung Cancer Patients

  • Ke, Hong-Gang;Li, Jun;Shen, Yi;You, Qing-Sheng;Yan, Yu;Dong, Han-Xuan;Liu, Jun-Hua;Shen, Zhen-Ya
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.9
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    • pp.4413-4416
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    • 2012
  • Aim: Individual differences in chemosensitivity and clinical outcome in non-small cell lung cancer (NSCLC) patients treatment with platinum-based chemotherapy may be due to genetic factors. Our study aimed to investigate the prognostic role of GSTP1, XRCC1 and XRCC3 in NSCLC patients treated with chemotherapy. Methods: A total of 460 cases were consecutively selected from The Affiliated Hospital of Nantong University between Jan. 2003 to Nov. 2006, and all were followed-up until Nov. 2011. Genotyping of GSTP1 Ile105Val, XRCC1 Arg194Trp, XRCC1 Arg399Gln and XRCC3 Thr241Met was conducted by duplex polymerase-chain-reaction with confronting-two-pair primer methods. Results: Patients with GSTP Val/Val exhibited a shorter survival time, and had a 1.89 fold greater risk of death than did those with the IIe/IIe genotype. For XRCC1 Arg194Trp, the variant genotype Trp/Trp was significantly associated with a decreased risk of death from NSCLC when compared with the Arg/Arg. Individuals carrying XRCC1 399Gln/Gln genotype had a longer survival time, with a lowered risk of death from NSCLC. Conclusion: This study indicated that GSTP1 Ile105Val, XRCC1 Arg194Trp and XRCC1Arg399Gln genes have a role in modifying the effect of platinum-based chemotherapy for NSCLC patients in a Chinese population. Our findings provide information for therapeutic decisions for individualized therapy in NSCLC cases.

Suppressed DNA Repair Mechanisms in Rheumatoid Arthritis

  • Lee, Sang-Heon;Firestein, Gary S
    • IMMUNE NETWORK
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    • v.2 no.4
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    • pp.208-216
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    • 2002
  • Background: Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity. Key members of the MMR system include MutS${\alpha}$ (comprised of hMSH2 and hMSH6), which can sense and repair single base mismatches and 8-oxoguanine, and MutS${\beta}$ (comprised of hMSH2 and hMSH3), which repairs longer insertion/deletion loops. Methods: To provide further evidence of DNA damage, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells (PBC) of RA patients using specific primer sequences for 5 key microsatellites. Results: Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis (OA) tissue. Western blot analysis of the same tissues for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Western blot analysis demonstrated constitutive expression of hMSH2, 3 and 6 in RA and OA FLS. When FLS were cultured with SNAP, the RA synovial pattern of MMR expression was reproduced (high hMSH3, low hMSH6). Conclusion: Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.

A Study on the Joint Property by the Surface Treatment Method on the Jointing Method of PET Film using the High Hardness Liquid (고경질 도막을 이용한 PET 필름 접합공법의 필름 표면처리 방법에 따른 접합특성 연구)

  • Lee, Jong-Suk;Kim, Young-Sam;Shin, Hong-Chol;Kim, Young-Geun;Kang, Chung-Mo
    • Journal of the Korea institute for structural maintenance and inspection
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    • v.18 no.3
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    • pp.153-159
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    • 2014
  • This research reviewed the joint and duration characteristics depending on the surface treatment condition and lap spliced length in the PET film jointing method using the high hardness liquid material. As a result, the corona discharge treatment was improved to the contact angle, joint tensile strength, and joint peel resistance compared to non-treatment. Particularly, a surface treatment E (Corona discharge + Primer + PU bond + Polyester fabric) turned out to the best, and especially when the lap spliced length is longer than 15mm, stable joint performance was secured under the long term deterioration treatment of 16 weeks. Thus, the joint is considered to be applicable as the water-proof material.

Induction of Somatic Embryos and RAPD Analysis in Regenerated Plantlets of Bupleurum falcatum L. (자호(紫胡)의 체세포배(體細胞胚) 형성(形成)과 재생(再生) 식물체(植物體)의 RAPD 분석(分析))

  • Park, Cheol-Ho;Yu, Chang-Yeon;Seo, Jeong-Sik;Kim, Ki-Sik;Ahn, Sang-Duek;Chang, Byoung-Ho
    • Korean Journal of Medicinal Crop Science
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    • v.3 no.1
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    • pp.50-55
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    • 1995
  • This study was conducted to determine the optimum conditions for induction and different somatic of somatic embryos as well as germination of encapsulated and stored somatic embryos. Somatic embryos was better formed in 1/2X MS medium than full - strength MS medium. 0.1 to 1.0mg/lBA and kinetin promoted shoot differentiation of somatic embryos. Higher concentration tend to inhibit differentiation. IAA affect positively both root and shoot growth. In vitro germination of somatic embryos encapsulated with 2% alginate matrix containing 1/2 MS nutrient medium and $AgNO_3$ 5mg/l was 86%. Storage of somatic embryos was effecive at $5^{\circ}C$ but the germination rate decreased with longer storage period. RAPD analyses with plants regenerated from the somatic embryos showed DNA polymorphism, indicating abolition of primer binding site by point mutation, deletion, or insertion of certain sequences.

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Nail DNA and Possible Biomarkers: A Pilot Study

  • Park, Joshua;Liang, Debbie;Kim, Jung-Woo;Luo, Yongjun;Huang, Taesheng;Kim, Soo-Young;Chang, Seong-Sil
    • Journal of Preventive Medicine and Public Health
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    • v.45 no.4
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    • pp.235-243
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    • 2012
  • Objectives: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples. Methods: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments. Results: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study. Conclusions: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

Association of A/T Rich Microsatellites with Responses to Artificial Selection for Larval Developmental Duration in the Silkworm Bombyx mori

  • Pradeep, Appukuttan Nair Retnabhavan;Awasthi, Arvind Kumar;Urs, Raje Siddaraje
    • Molecules and Cells
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    • v.25 no.4
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    • pp.467-478
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    • 2008
  • Simple sequence repeats (SSRs) and interSSR (ISSR) marker systems were used in this study to reveal genetic changes induced by artificial selection for short/long larval duration in the tropical strain Nistari of the silkworm Bombyx mori. Artificial selection separated longer larval duration (LLD) ($29.428{\pm}0.723days$) and shorter larval duration (SLD) ($22.573{\pm}0.839days$) lines from a base, inbred population of Nistari (larval span of $23.143{\pm}0.35days$). SSR polymorphism was observed between the LLD and SLD lines at one microsatellite locus, Bmsat106 ($CA_7$) and at two loci of 1074 bp and 823 bp generated with the ISSR primer UBC873. Each of these loci was present only in the LLD line. The loci segregated in the third generation of selection and were fixed in opposite directions. In the $F_2$ generation of the $LLD{\times}SLD$ lines, the alleles of Bmsat106 and $UBC873_{1074bp}$ segregated in a 1:1 ratio and the loci were present only in the LLD individuals. $UBC873_{823bp}$ was homozygous. Single factor ANOVA showed a significant association between the segregating loci and longer larval duration. Together, the two alleles contributed to an 18% increase in larval duration. The nucleotide sequences of the $UBC873_{1074bp}$ and $UBC873_{823bp}$ loci had 67% A/T content and consisted of direct, reverse, complementary and palindromic repeats. The repeats appeared to be "nested" (59%) in larger repeats or as clustered elements adjacent to other repeats. Of 203 microsatellites identified, dinucleotides (67.8%) predominated and were rich in A/T and T/A motifs. The sequences of the $UBC873_{1074bp}$ and $UBC873_{823bp}$ loci showed similarity (E = 0.0) to contigs located in Scaffold 010774 and Scaffold 000139, respectively, of the B. mori genome. BLASTN analysis of the $UBC873_{1074bp}$ sequence showed significant homology of (nt.) 45-122 with upstream region of three exons from Bombyx. The complete sequence of this locus showed ~49% nucleotide conservation with transposon 412 of Drosophila melanogaster and the Ikirara insertions of Anopheles gambiae. The A + T richness and lack of coding potential of these small loci, and their absence in the SLD line, reflect the active process of genetic change associated with the switch to short larval duration as an adaptation to the tropics.

Cloning, cSNP Identification, and Genotyping of Pig Complement Factor B(CFB) Gene Located on the SLA Class III Region (SLA Class III 영역의 돼지 Complement Factor B(CFB) 유전자의 Cloning, cSNP 동정 및 유전자형 분석)

  • Kim, Jae-Hwan;Lim, Hyun-Tae;Seo, Bo-Yeong;Zhong, Tao;Yoo, Chae-Kyoung;Jung, Eun-Ji;Jeon, Jin-Tae
    • Journal of Animal Science and Technology
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    • v.50 no.6
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    • pp.753-762
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    • 2008
  • The primers for RT-PCR and RACE-PCR were designed by aligning the pig genomic sequence and the human complement factor B(CFB) coding sequence(CDS) from the GenBank. Each PCR product was amplified in pig cDNA and sequencing was carried out. The CDS length of pig CFB gene was determined to be 2298 bp. In addition, the pig CDS was more longer than human and mouse orthologs because of insertion and deletion. The identities of porcine nucleotide sequences with those of human and mice were 84% and 80%, and the identities of amino acids were 79% to 77%, respectively. Three complement control protein(CCP) domains, one Von Willebrand factor A(VWFA) domain and a serine protease domain, that are revealed typically in mammals, were found in the pig CFB gene. Based on the CDSs determined, the primers were designed in intron regions for amplification of entire length of exons. In amplification and direct sequencing with genomic DNAs of six pig breeds, three cSNPs(coding single nucleotide polymorphisms) were identified and verified as missense mutations. Using the Multiplex-ARMS method, we genotyped and verified the mutations identified from direct sequencing. To demonstrate recrudescence, we performed both direct sequencing and Multiplex-ARMS with two randomly selected DNA samples. The genotype of each sample exhibited the same results using both methods. Therefore, three cSNPs were identified from pig CFB gene and that can be used for haplotype analysis of the swine leukocyte antigen(SLA) class III region. Moreover, the results indicate that the Multiplex-ARMS method should be powerful for genotyping of genes in the SLA region.

Development and Genetic Diversity Analysis of Microsatellite Markers Using Next-generation Sequencing in Seriola quinqueradiata (차세대 염기서열 분석법을 이용한 방어(Seriola quinqueradiata)의 microsatellite 마커의 개발 및 유전적 특성 분석)

  • Dong, Chun Mae;Lee, Mi-Nan;Kim, Eun-Mi;Park, Jung Youn;Kim, Gun-Do;Noh, Jae Koo
    • Journal of Life Science
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    • v.30 no.3
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    • pp.291-297
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    • 2020
  • This study was conducted to develop microsatellite markers in Seriola quinqueradiata using next-generation sequencing. A total of 28,873,374 reads were generated on an Illumina Hiseq2500 system, yielding 7,247,216,874 bp sequences. The de novo assembly resulted in 466,359 contigs. A total of 132 contigs (0.43%), including 60 microsatellite loci, were derived from 30,729 contigs longer than 518 bp. A total of 60 primer sets were designed from the 132 microsatellite loci. A total of 15 polymorphic nuclear microsatellite loci were chosen to evaluate population genetic parameters in the parents and offspring. The mean number of effective alleles was 18.5, ranging from 11 to 30. The observed heterozygosity (HO) and expected heterozygosity (HE) ranged between 0.431 and 0.972 with an average of 0.812 and from 0.782 to 0.949 with an average of 0.896, respectively. No significant linkage disequilibrium was observed after Bonferroni revision in any loci. The results show that the 15 polymorphic nuclear microsatellite markers can be used to study the population and conservation genetics of S. quinqueradiata in Korea. To ensure the success of artificial seedling production technology, genetic variations between the parent and offspring populations should be monitored, and inbreeding should be controlled.

Characteristics of a new mid-high temperature adaptable oyster mushroom variety 「Gonji-5ho」 for bag culture (중고온성 봉지재배용 신품종 느타리 『곤지5호』 육성 및 특성)

  • Choi, Jong-In;Ha, Tai-Moon;Jeon, Dae-Hoon;Ju, Young-Cheul;Cheong, Jong-Chun
    • Journal of Mushroom
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    • v.10 no.3
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    • pp.115-119
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    • 2012
  • 'Gonji-5ho', a new variety of oyster mushroom, for the bag culture, was bred by mating two monokaryons isolated from 'Chiak-3ho'and 'Suhan-1ho'. In the characteristics of fruit body, pilei were thick and gray and stipes were thick, and long, and soft. It was better in elasticity and cohesivness of tissue compared to Suhan-1ho. Compared to other varieties, it was suitable to grow at higher temperature. The range of optimum temperature for the mycelial growth was around 26~29 and that for the pinheading and growth of fruit body was around $18{\sim}20^{\circ}C$. But when it was cultuered in lower than $15^{\circ}C$, growth was not uniform, culture period was longer, and stipes were uneven. In the bag culture, it was required around 18 days in incubation period and 3 days in primordia formation. The fruit body growth was vital and uniform. The yield was 221.4g/1kg bag.

RESIN TAG FORMATION OF SELF-ETCHING ADHESIVES (자가부식 접착제의 레진 Tag 형성)

  • Kim, Young-Jae;Jang, Ki-Taeg
    • Journal of the korean academy of Pediatric Dentistry
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    • v.30 no.1
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    • pp.143-152
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    • 2003
  • The aims of the present study was to observe resin tag of the resin/enamel, dentin interface produced by self-etching adhesive systems and evaluate effect of additional acid etching on resin tag formation. Three self-etching primer(SE bond, AQ bond and L Pop) and an one bottle adhesive(Single bond) were used. Flat occlusal enamel and dentin disks were obtained from extracted human molars. A total of 20 surfaces were collected and divided into four groups of 5 samples. One-half of each specimen in each group was etched with 35% phosphoric acid prior to the application of each adhesive system, with the second half being kept unetched. Subsequently, resin composite was placed and polymerized. The samples were sliced and immersed into HCl and NaOCl solutions, followed by drying and sputter coating for examination with a SEM. The results were as follows; 1. Additional etching side of dentin displayed longer and thicker resin tag than unetched side in all self-etching adhesive groups. 2. In enamel, additional etching side displayed deeper and more distinct etching pattern than unetched side except L Pop. There is no difference between etched and unetched enamel in L Pop. The results obtained suggest the self-etching adhesive did not etch enamel and penetrate into dentinal tubule as deeply as did additional etching. Further research should include the evaluation of the relationship of boding strength, microleakage and resin tag morphology.

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