• Title/Summary/Keyword: t-RFLP

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Rapid Detection of H-RAS Point Mutation Using Two-Step Polymerase Chain Reaction-Restriction Fragment Length Polymorphism

  • Park, Young-Suk;Lee, Kyung-Ok;Chai, Young-Gyu
    • BMB Reports
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    • v.29 no.5
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    • pp.442-447
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    • 1996
  • Mutations in codon 12, 13 and 61 of one of the three ras genes, H-ras, K-ras and N-ras, convert these genes into active oncogenes. The presence of H-ras gene mutations have important prognostic implications in various cancers. In this study, the H-ras gene mutations were investigated by two-step PCRRFLP in patients with bladder and stomach cancer. For the control experiments, T24 and SK2 cell lines were used. In a total of 36 bladder cancer patient cases, five (13.9%) mutations were found by this method. Of these, point 12 mutations were two (5.6%) cases and point 61 mutations were three (8.3%) cases. On the other hand, H-ras mutation was not found in 29 cases of stomach cancer. The results of the mutated H-ras gene confirmed by direct sequencing analysis were correlated well with PCR analysis. From the sensitivity test, the H-ras mutation was found to have about 0.2% of mutated DNA mingled in normal DNA. In conclusion, the H-ras mutation has a higher clinical Significance in bladder cancer than stomach cancer. Moreover the two-step PCR-RFLP method is sensitive, rapid and relatively simple for clinical work in detecting H-ras point mutations.

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Identification of Quantitative Trait Loci Associated with Traits of Soybean for Sprout

  • Lee, Suk-Ha;Park, Keum-Yong;Lee, Hong-Suk;H. Roger Boerma
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.44 no.2
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    • pp.166-170
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    • 1999
  • The identification of quantitative trait loci (QTL) has the potential to enhance the efficiency of im- proving food processing traits of soybean. In this study, 92 restriction fragment length polymorphism (RFLP) loci and two morphological markers (W$_1$ and T) were used to identify QTL associated with food processing traits of soybean for sprout in 83 F$_2$-derived lines from a cross of 'Pureun' x 'Jinpum 2'. The genetic map consisted of 76 loci which covered about 760 cM and converged into 20 linkage groups. Eighteen markers remained unlinked. Phenotypic data were collected for hypocotyl length, abnormal seedling rate, and sprout yield seven days after seed germination at 2$0^{\circ}C$. Based on the single-factor analysis of variance, eight independent markers were associated with hypocotyl length. Four of seven markers associated with abnormal seedling rate were identified as independent. Seven loci were associated with sprout yield. For three different traits, much of genetic variation was explained by the identified QTL in this population. Several RFLP markers in linkage group (LG) Bl were detected as being associated with three traits, providing a genetic explanation for the biological correlation of sprout yield with hypocotyl length (r=OA07***) and with abnormal seedling rate (r=-406***).

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Transforming growth factor-β gene promoter polymorphism : its association with renal involvement in Henoch-Schölein Purpura in childhood (소아 Henoch-Schölein purpura에서 전환성장인자-β 프로모터유전자의 유전학적 다형성과 신장침범의 관련성)

  • Lee, Seung Ho;Jee, Hwa Young;Kim, Hwang Min;Yeh, Byung Il
    • Clinical and Experimental Pediatrics
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    • v.51 no.5
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    • pp.523-527
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    • 2008
  • Purpose : Several cytokines play important roles in the inflammatory process of Henoch-$Sch\ddot{o}lein$ Purpura (HSP). It is likely that transforming growth $factor-{\beta}$ ($TGF-{\beta}$) is involved in the pathogenesis of HSP. The purpose of this study is to investigate whether $TGF-{\beta}$ promoter polymorphism is associated with the renal involvement of childhood HSP. Methods : Thirty-four patients younger than 15 years, who had been diagnosed with HSP, as well as 27 controls, were examined. Patients and controls were genotyped for $TGF-{\beta}$ C-509T by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results : The T allelic frequencies in patients and controls showed no difference (45% vs. 48.8%). No allele or genotype differences between the group of HSP group and control group were observed. The frequencies of $TGF-{\beta}$ 509 genotypes TT, TC, and CC were no different between patients and controls (26% vs. 22%). The TT genotype of polymorphism of the $TGF-{\beta}$ C-509T gene had no relation to the susceptibility of children to HSP and renal involvement in HSP. Conclusion : $TGF-{\beta}$ T allele may not be related to the susceptibility of children to HSP. The TT genotype of polymorphism of the $TGF-{\beta}$ C 509T gene does not appear to have an influence on renal involvement in childhood HSP.

A study of Association of the H-FABP RFLP with Economic Traits of Pigs (돼지 H-FABP 유전자의 다형성 및 경제 형질과의 연관성 구명)

  • Choi, B.H.;Kim, T.H.;Lee, J.W.;Cho, Y.M.;Lee, H.Y.;Cho, B.W.;Cheong, I.C.
    • Journal of Animal Science and Technology
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    • v.45 no.5
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    • pp.703-710
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    • 2003
  • The purpose of this study was to detect association between genetic variation and economic trait in the porcine heart type fatty acid-binding protein gene as a candidate gene for the traits related with growth and meat quality in pigs. The H-FABP is a 15-kDa protein expressed in several tissues with high demand for fat metabolism such as cardiac and skeletal muscle and lactating mammary gland. H-FABP is small intracellular protein involved in fatty acid transport from the plasma membrane to the site of $\beta$-oxidation and/or triacylglycerol or phospholipid synthesis. In this study, H-FABP PCR-RFLP was performed in F$_2$ population composed of 214 individuals from an intercross between Korean Native Boars and Landrace sows. PCR products from two primer sets within H-FABP gene were amplified in 850bp and 700bp. Digestion of PCR products with the restriction digestion enzymes HaeⅢ and HinfⅠ, revealed fragment length polymorphisms(RFLPs). The genotype frequencies from H-FABP/HaeⅢ was .29 for genotype DD, .53 for genotype Dd, and .15 for genotype dd, respectively. The genotype frequencies of HH, Hh, and hh from H-FABP/HinfⅠ was .38, .41 and .20, respectively, in the population. Relationships between their genotypes and economic traits were estimated. In H-FABP/HaeⅢ locus, there were specific genotypes(Dd and dd) associated with economic traits such as body weights at 3, 5, 12, and 30 week of age (p〈.05 to .001). The ‘d’ allele was associated with gaining of body weight. In H-FABP/HinfⅠ locus, Genotypes of HH and Hh associated with growth traits such as body weights at 5, 12, and 30 week of age (p〈.05 or p〈.001) and back fat thickness, body fat including abdominal and trimmed fat (p〈.001) and intramuscular fat(p〈.05) The ‘H’ allele was positively associated with gaining of body weight and fatness deposition. In conclusion, a significant association of the H-FABP gene from its genetic variation was found on body weight, intramuscular fat and backfat thickness.

PCR and RFLP-based CYP2D6(B) and CYP2D6(T) Genotyping for Korean Lung Cancer Cases and Controls (한국인 폐암환자와 대조군의 CYP2D6 유전적 다형성에 관한 연구)

  • Chun, Jin-Ho;Lee, Chang-Hee;Urm, Sang-Hwa;Son, Byung-Chul;Park, Jun-Han;Jung, Kui-Oak;Sohn, Chang-Hak;Yoon, Hye-Kyoung;Son, Choon-Hee;Kim, Hyung-In;Jeong, Jin-Sook
    • Journal of Preventive Medicine and Public Health
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    • v.31 no.1 s.60
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    • pp.1-14
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    • 1998
  • The genetically determined CYP2D6 activity as considered to be associated with cancer susceptibility with inter-individual variation. Genetic polymorphism of CYP2D6(B) and CYP2D6(T) was determined by the two polymerase chain reaction(PCR) and BstN1 and EcoN1 restriction fragment length polymorphisms(RFLP) for 67 lung cancer cases and 95 healthy volunteer controls. The cases were composed of 26 squamous cell carcinoma, 14 small cell carcinoma, 10 adenocarcinoma, 3 large cell undifferentiated carcinoma, and 14 not histologically diagnosed. The results were gained from the 142 subjects (57 cases and 85 controls) who observed successfully in two PCR and BstNl/EcoN1 RELP. Only one and no mutant allele of the CYP2D6(B) and CYP2D6(T) gene was detected, that is, the frequency of mutant allele was very low; 0.7%(1/142) and 0%(0/142), respectively. Detected mutant allele of the CYP2D6(B) was beterozygous type(WM). The odds ratios for lung cancer susceptibility with CYP2D6(B) and CYP2D6(T) genotype were not calculated. These results are similar to the previous understanding that the mutant allele is very rare in Orientals compared to Caucasians, therefore, it considered that CYP2D6(B) and CYP2D6(T) genotypes have maybe no association with lung cancer susceptibility in Koreans. This is the basic data of CYP2D6(B) and CYP2D6(T) genotypes for Koreans. It would be hepful for further study to determine lung cancer susceptibility of Koreans with the data about CYP1A1, CYP2E1, GSTM1 from future study.

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The Association between A-FABP Promoter Genotype and Carcass Traits in Pigs (비육돈의 도체형질과 A-FABP Promoter 유전자형의 연관 분석)

  • Han, Sang-Hyun;Cho, In-Cheol;Lee, Chong-Eon;Ko, Moon-Suck;Seong, Pil-Nam;Park, Beom-Young
    • Food Science of Animal Resources
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    • v.27 no.3
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    • pp.382-386
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    • 2007
  • The association between the promoter region genotypes of the porcine adipocyte fatty acid binding protein (A-FABP) gene and carcass traits in commercial pigs was examined. Interestingly, only two different genotypes (-406T/T and T/C) for the A-FABP gene were detected using Hinf-RFLP for the substitution mutation T-406C in the A-FABP promoter in commercial pigs, and no -406C/C homozygotes were detected. The reason for the lack of the -406 C/C genotype is due to the breeding system in which only Duroc, which has a high frequency of -406C/- in this locus among the three breeds involved in commercial pig production, is typically used as a terminal sire. The pigs containing the genotype -406C/- were significantly associated with an increase in intramuscula. fat content and carcass weight (p<0.05), but there was no association with the other carcass traits tested (fat composition, color score, texture score, moisture, and separation score between muscles). This study suggests that the -406C/- genotype of the porcine A-FABP gene may not only be a useful molecular marker for intramuscular fat, but may also contribute to the improvement of meat quality by the production of well-marbled pigs by breeding animals containing this genotype, especially Duroc, as a terminal sire for commercial pigs.

Influence of Elevated $CO_2$ on Denitrifying Bacterial Community in a Wetland Soil (이산화탄소 증가가 습지토양의 탈질세균 군집구조에 미치는 영향)

  • Lee Seung-Hoon;Kim Seonyoung;Kang Hojeong
    • Korean Journal of Microbiology
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    • v.40 no.3
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    • pp.244-247
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    • 2004
  • To investigate the effects of elevated $CO_2$ on the denitrifying bacterial community structure in a wetland soil, dynamics of bacterial community structure was explored in an artificial wetland ecosystem with one of three plant species (T. latifolia, S. lacustris, and 1. effusus) under two levels of $CO_2$(370 ppm or 740 ppm) after 110day incubation. For the analysis of bacterial community structure, functional genes such as nitrite reductase genes (nirS) were PCR-amplified followed by cloning of PCR products and screening by restriction fragment length polymorphism (RFLP). nirS gene fragments were amplified in all analyzed soil samples. Species richness estimated by the number of distinct phylotypes were 83 and 95 in the ambient $CO_2$ treatment and the elevated treatment, respectively. Two phylotypes (type 1 and type 2) were dominant in both of the treatments. Elevated $CO_2$ treatment increased species richness of denitrifying as well as changed a large proportion of denitrifier phylotypes compared to those of the ambient treatment. Overall, the data in this study suggested that the denitrifying communities in the wetland soil are diverse and that the richness of denitrifying bacterial community might be affected by elevated $CO_2$ treatment.

DNA Polymorphism in SLC11A1 Gene and its Association with Brucellosis Resistance in Indian Zebu (Bos indicus) and Crossbred (Bos indicus×Bos taurus) Cattle

  • Kumar, Nishant;Ganguly, Indrajit;Singh, Rajendra;Deb, Sitangsu M.;Kumar, Subodh;Sharma, Arjava;Mitra, Abhijit
    • Asian-Australasian Journal of Animal Sciences
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    • v.24 no.7
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    • pp.898-904
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    • 2011
  • The PCR- restriction fragment length polymorphism (RFLP) in and around TM4 of SLC11A1 gene and its association with the incidences of brucellosis in Hariana breed (Bos indicus) and Holstein Friesian crossbred (Bos indicus${\times}$Bos taurus) cattle was examined. A fragment of 954 bp encoding the TM4 was amplified, and RFLP was identified by digestion of the amplicon independently with AluI and TaqI. The amplicon (GenBank Acc. No. AY338470 and AY338471) comprised of a part of exon V (<59 bp) and VII (62>), and entire intron 5 (423 bp), exon VI (71 bp) and intron 6 (339 bp). Digestion with AluI revealed the presence of two alleles viz, A (281, 255, 79 and 51 bp) and B (541, 255, 79 and 51 bp). The frequency of A allele was estimated as 0.80 and 0.73 in Hariana and crossbred cattle, respectively. Due to presence of a polymorphic TaqI site at intron 5, two alleles: T (552 and 402 bp) and Q (231, 321 and 402 bp) were identified. The frequency of T allele was estimated as 0.96 and 0.97, respectively. For association study, on the basis of serological tests and history of abortion, the animals were grouped into "affected" and "non-affected". However, no association could be established with the observed RFLPs.

Presence and Growth of Ammonia-oxidizing Bacteria in Anaerobic Ammonium Oxidation Enrichment (아나목스 농후배양에서 암모니아 산화균의 자생 특성)

  • Bae, Hyokwan;Paul, Tanusree;Jung, Jin-Young
    • Journal of Korean Society on Water Environment
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    • v.36 no.3
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    • pp.220-228
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    • 2020
  • Anaerobic ammonium oxidation (AMX) is a cost-efficient biological nitrogen removal process. The coexistence of ammonia-oxidizing bacteria (AOB) in an AMX reactor is an interesting research topic as a nitrogen-related bacterial consortium. In this study, a sequencing batch reactor for AMX (AMX-SBR) was operated with a conventional activated sludge. The AOB in an AMX bioreactor were identified and quantified using terminal restriction fragment length polymorphism (T-RFLP) and real-time qPCR. A T-RFLP assay based on the ammonia monooxygenase subunit A (amoA) gene sequences showed the presence of Nitrosomonas europaea-like AOB in the AMX-SBR. A phylogenetic tree based on the sequenced amoA gene showed that AOB were affiliated with the Nitrosomonas europaea/mobilis cluster. Throughout the enrichment period, the AOB population was stable with predominant Nitrosomonas europaea-like AOB. Two OTUs of amoA_SBR_JJY_20 (FJ577843) and amoA_SBR_JJY_9 (FJ577849) are similar to the clones from AMX-related environments. Real-time qPCR was used to quantify AOB populations over time. Interestingly, the exponential growth of AOB populations was observed during the substrate inhibition of the AMX bacteria. The specific growth rate of AOB under anaerobic conditions was only 0.111 d-1. The growth property of Nitrosomonas europaea-like AOB may provide fundamental information about the metabolic relationship between the AMX bacteria and AOB.

Effects of elevated CO2 on organic matter decomposition capacities and community structure of sulfate-reducing bacteria in salt marsh sediment

  • Jung, Soo-Hyun;Lee, Seung-Hoon;Park, Seok-Soon;Kang, Ho-Jeong
    • Journal of Ecology and Environment
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    • v.33 no.3
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    • pp.261-270
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    • 2010
  • Increasing atmospheric $CO_2$ affects the soil carbon cycle by influencing microbial activity and the carbon pool. In this study, the effects of elevated $CO_2$ on extracellular enzyme activities (EEA; ${\beta}$-glucosidase, N-acetylglucosaminidase, aminopeptidase) in salt marsh sediment vegetated with Suaeda japonica were assessed under ambient atmospheric $CO_2$ concentration (380 ppm) or elevated $CO_2$ concentration (760 ppm) conditions. Additionally, the community structure of sulfate-reducing bacteria (SRB) was analyzed via terminal restriction fragments length polymorphism (T-RFLP). Sediment with S. japonica samples were collected from the Hwangsando intertidal flat in May 2005, and placed in small pots (diameter 6 cm, height 10 cm). The pots were incubated for 60 days in a growth chamber under two different $CO_2$ concentration conditions. Sediment samples for all measurements were subdivided into two parts: surface (0-2 cm) and rhizome (4-6 cm) soils. No significant differences were detected in EEA with different $CO_2$ treatments in the surface and rhizome soils. However, the ratio of ${\beta}$-glucosidase activity to N-acetylglucosaminidase activity in rhizome soil was significantly lower (P < 0.01) at 760 ppm $CO_2$ than at 380 ppm $CO_2$, thereby suggesting that the contribution of fungi to the decomposition of soil organic matter might in some cases prove larger than that of bacteria. Community structures of SRB were separated according to different $CO_2$ treatments, suggesting that elevated $CO_2$ may affect the carbon and sulfur cycle in salt marshes.