• Title/Summary/Keyword: PCR-based Markers

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Association of Poor Prognosis Subtypes of Breast Cancer with Estrogen Receptor Alpha Methylation in Iranian Women

  • Izadi, Pantea;Noruzinia, Mehrdad;Fereidooni, Foruzandeh;Nateghi, Mohammad Reza
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.8
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    • pp.4113-4117
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    • 2012
  • Breast cancer is a prevalent heterogeneous malignant disease. Gene expression profiling by DNA microarray can classify breast tumors into five different molecular subtypes: luminal A, luminal B, HER-2, basal and normal-like which have differing prognosis. Recently it has been shown that immunohistochemistry (IHC) markers including estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2), can divide tumors to main subtypes: luminal A (ER+; PR+/-; HER-2-), luminal B (ER+;PR+/-; HER-2+), basal-like (ER-;PR-;HER2-) and Her2+ (ER-; PR-; HER-2+). Some subtypes such as basal-like subtype have been characterized by poor prognosis and reduced overall survival. Due to the importance of the ER signaling pathway in mammary cell proliferation; it appears that epigenetic changes in the $ER{\alpha}$ gene as a central component of this pathway, may contribute to prognostic prediction. Thus this study aimed to clarify the correlation of different IHC-based subtypes of breast tumors with $ER{\alpha}$ methylation in Iranian breast cancer patients. For this purpose one hundred fresh breast tumors obtained by surgical resection underwent DNA extraction for assessment of their ER methylation status by methylation specific PCR (MSP). These tumors were classified into main subtypes according to IHC markers and data were collected on pathological features of the patients. $ER{\alpha}$ methylation was found in 25 of 28 (89.3%) basal tumors, 21 of 24 (87.5%) Her2+ tumors, 18 of 34 (52.9%) luminal A tumors and 7 of 14 (50%) luminal B tumors. A strong correlation was found between $ER{\alpha}$ methylation and poor prognosis tumor subtypes (basal and Her2+) in patients (P<0.001). Our findings show that $ER{\alpha}$ methylation is correlated with poor prognosis subtypes of breast tumors in Iranian patients and may play an important role in pathogenesis of the more aggressive breast tumors.

A taxonomic study of Abeliophyllum Nakai (O1eaceae) based on RAPD analysis (RAPD분석에 의한 미선나무속의 분류학적 연구)

  • 김동갑;박경량;김주환
    • Korean Journal of Plant Resources
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    • v.15 no.1
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    • pp.26-35
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    • 2002
  • Abeliophyllum distichum Nakai (O1eaceae) is a monotype of Korea and is distinguished from related genus Forsythia Vahl by the morphological characters such as fruit shape, flower color and etc. Even though several intraspecific taxa were reported according to the color of flowers and shape of fruits, there have been many controversals on the taxonomic indentity and status of rank on those taxa. In the present study, we performed the RAPD analysis to delimit the infraspecific taxa of Abeliophyllum distichum and to investigate the genetic polymorphism and relationships among 12 populations. 212 scorable RAPD markers with 70 common markers were found from the PCR reactions with 16 random oligoprimers and were analyzed by Nei's genetic distance. From 0.108 to 0.321 of genetic variations were showed among the taxa. Some regional groups instead of same taxa were clustered from the phonogram of UPGMA analysis. Also, we could not find distinct lineage among intraspecific taxa. The result from RAPD analysis supported that the infraspecific taxa of Abeliophyllum distichum might be the individual variations and treated as the same taxa. RAPD analysis was very useful to confirm the high gene pool with diverse genetic polymorphism among Abeliophyllum distichum populations.

Genetic Diversity and Phylogenetic Relationship of Genus Phyllostachys by ISSR Markers (ISSR 분자 마커를 이용한 왕대속 대나무의 유전적 다양성 및 계통 관계)

  • Lee, Song-Jin;Huh, Man-Kyu;Huh, Hong-Wook
    • Journal of Life Science
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    • v.17 no.11
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    • pp.1482-1487
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    • 2007
  • Four taxa of the genus Phyllostachys were analysed with PCR-based molecular inter-simple sequence repeat (ISSR) determine markers to determine their phylogenetic relationships. Many species of this genus are regarded as economical and ecologically important in the world. With the nine primers screened, ISSR assay generated a total of 64 reproducible bands. Analysis of ISSR from individual plants of genus Phyllostachys resulted in 43 polymorphic bands with 70.49%. When species were grouped by four taxa, within group diversity was 0.092 (Hs), while among group diversify was 0.499 (Gst) on a per locus basis. The estimated gene flow (Nm) between the pairs of species was 0.559. Hence, we can expect weak or low gene flow among species. Total mean genetic diversity ($H_T$) for four taxa was 0.291. The ISSR data allowed us to resolve well-supported clades in the genus Phyllostachys. The four taxa of the genus Phyllostachys analyzed were distinctly related to a monophyletic.

DNA Polymorphism and Assessments of Genetic Relationships in genus Zoysia Based on Simple Sequence Repeat Markers (ISSR에 의한 잔디속 식물의 DNA 다형성과 유전적 관계 평가)

  • Huh, Man Kyu
    • Journal of Life Science
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    • v.25 no.3
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    • pp.257-262
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    • 2015
  • The genetic variability of four species of the genus Zoysia collected from South Korea was analyzed using an inter-simple sequence repeat (ISSR) marker system. Polymerase chain reactions (PCR) with eight ISSR primers generated 86 amplicons, 76 (87.1%) of which were polymorphisms. The polymorphism information content (PIC) value of the ISSR marker system was 0.848. The percentage of polymorphic loci (Pp) ranged from 41.2% to 44.7%. Nei’s gene diversity (H) ranged from 0.149 to 0.186, with an average overall value of 0.170. The mean of Shannon’s information index (I) value was 0.250. Total genetic diversity values (HT) varied between 0.356 (ISSR-1) and 0.418 (ISSR-16), for an average overall polymorphic loci of 0.345. Interlocus variation in within-species genetic diversity (HS) was low (0.170). On a per-locus basis, the proportion of total genetic variation due to differences among species (GST) was 0.601. This indicated that about 60.1% of the total variation was among species. Thus, about 39.9 of genetic variation was within species. The estimate of gene flow, based on GST, was very low among species of the genus Zoysia (Nm = 0.332). The phylogenic tree showed three distinct groups: Z. macrostachya and Z. tenuifolia clades and other species were formed the separated clusters. In conclusion, the ISSR assay was useful for detecting genetic variation in the genus Zoysia, and its discriminatory power was comparable to that of other genotyping tools.

Effects of enamel matrix derivative and titanium on the proliferation and differentiation of osteoblasts (법랑기질유도체를 도포한 타이태늄 표면에서 조골세포의 증식 및 분화)

  • Park, Sang-Hyun;Lee, In-Kyeong;Yang, Seung-Min;Shin, Seung-Yun;Lee, Yong-Moo;Ku, Young;Rhyu, In-Chul;Chung, Chong-Pyoung;Han, Soo-Boo;Choi, Sang-Mook
    • Journal of Periodontal and Implant Science
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    • v.33 no.3
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    • pp.359-372
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    • 2003
  • Among objectives of periodontal therapy. the principal one is the morphological and functional reconstruction of lost periodontal supporting tissues. This includes de novo formation of connective tissue attachment and the regrowth of alveolar bone. The use of enamel matrix derivative(EMD) may be a suitable means of regeneration new periodontal attachment in the infrabony defects. Implant used to replace lost tooth but, implantitis occurred after installation. The purpose of this study was to investigate the effects of EMD on differentiation and growth of osteoblast in titanium disc. Twentyfive millimeter diameter and 1mm thick Ti disc which was coated 25, 50, 100, 200${\mu}g$/ml of EMD(Emdogain(R)) used as experimental group, 25, 50, 100, 200ng/d of rhBMP-2 as positive control group, and no coat as negative control group. A human osteosarcoma cell line Saos-2 was cultured in Ti disc and cell proliferation and Alkaline phosphatase (ALP) activity were measured at 1 and 6 days. PCR was performed at 2 and 8 hours. Semi-quantitative RT-PCR for mRNA expressions of various osteoblastic differentiation markers -type I collagen, ALP, osteopontin, and bone sialoprotein - were performed at appropriate concentrations based upon the results of MTT and ALP assay. Cultured cell-disc complexes were prepared for scanning electron microscopy (SEM) at 2 hour. Data were analyzed using Mann-Whitney and repeated- measures 1-way analysis of variance(SPSS software version 10,SPSS. Chicago. IL). After culture, there was more osteoblast in EMD100${\mu}g$/ml than in EMD50, 200${\mu}g$/ml on day 6. There was significant difference in experimental and positive control group compared control group, as times go by(1 and 6 days). Alkaline phosphatase activity was different significantly in EMD100, 200${\mu}g$/ml and BMP100, 200${\mu}g$/ml on day 6. The results of reverse transcriptase-polymerase chain reaction (RT-PCR) showed that expression of mRNA for ALPase, collagen type I, osteopontin. hone sialoprotein and BMP-2 was detected at 2 hour and 8 hour in EMI 200${\mu}g$/ml subgroup and BMP100ng/ml subgroup. The results of this study suggest that application of enamel matrix derivative on osteoblast attached to titanium surface facilitate the expression of bone specific protein and the differentiation and growth of osteoblast.

Cloning and Activity Analysis of the FosB Promoter Region from Human Genomic DNA (사람 핵DNA로부터 FosB 유전자 프로모터 클로닝 및 활성도 분석)

  • Na, Han-Heom;Kang, Yoonsung;Kim, Keun-Cheol
    • Journal of Life Science
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    • v.27 no.8
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    • pp.857-863
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    • 2017
  • The FBJ murine osteosarcoma viral oncogene homolog B (FosB) gene is located at chromosome 19, and encodes 43 Kda protein. Functionally, the FosB gene is important for differentiation, development, and pathogenesis. Furthermore, the FosB gene is suggested as possible biomarker for tracing disease prognosis. In this study, we constructed plasmid containing a FosB promoter region and evaluate its promoter activity. We analyzed the putative promoter region in FosB genomic DNA using bioinformatics program, and we found important regulatory elements in 1 Kb upstream from transcription start site (TSS). Therefore, we performed polymerase chain reaction (PCR) amplification on region from-1,555 upstream to +73 of the FosB genomic DNA, and PCR product was inserted into TA vector to create the $TA-1^{st}FosBp$ plasmid. We then prepared the primer sets, which contain a restriction enzyme site for Kpn1 and Nhe1, in order to reinsert into the TA vector to prepare $TA-2^{nd}FosBp$ plasmid. It was finally subcloned into pGL3-luc vector after enzyme cutting. To evaluate whether the cloned plasmid is useful in cell based experiment, we performed luciferase assay with pGL3-FosBp-luctransfection. FosB promoter activity was increased compared to empty vector, and this activity was significantly increased by treatment of doxorubicin and taxol. We obtained consistent data on regulation of FosB gene expression after anticancer drug treatment using Western blot analysis. The results suggest that promoter cloning of the human FosB gene is very useful for studying gene expression and analyzing biomarkers.

Cytomegalovirus Infection in Infantile Hepatitis

  • Na, So Young
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.15 no.2
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    • pp.91-99
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    • 2012
  • Purpose: The aims of this study was to compare and evaluate the clinical characteristics, laboratory data, and prognosis for infants under age 1 year with CMV hepatitis and those with viral hepatitis of unknown etiology. Methods: A retrospective study was conducted of infants under age 1 year who were admitted with acute hepatitis. The exclusion criteria consisted of: autoimmune, genetic, metabolic, toxic, HAV, HBV, HCV, toxoplasma, rubella, herpes simplex, and Epstein-Barr virus. The 30 patients included were divided into two groups based on markers for CMV (IgM anti-CMV, CMV PCR in urine, CMV culture in urine). Results: The median age of patients (n=15) was 2.8 months. No other organ involvement was detected in any patient. Peak serum total bilirubin levels (n=4) ranged from 2.6 to 6.7 mg/dL. Peak serum ALT levels ranged from 51 to 1,581 IU/L. The duration of ALT elevation ranged from 1.5 weeks to 26 weeks (median 9 weeks). All had recovered in full without ganciclovir; there were no cases of hearing loss. The median age of controls (n=15) was 2.5 months. Peak serum total bilirubin levels (n=4) ranged from 1.6 to 9.1 mg/dL. Peak serum ALT levels ranged from 26 to 1,794 IU/L. No significant differences were observed between both groups regarding the peak serum ALT levels, peak serum total bilirubin levels, duration of hyperbilirubinemia and ALT elevation. Conclusion: Although it was not possible to differentiate congenital infection with perinatal infection in this study, the prognosis of patients with CMV hepatitis without other organ involvement was good without ganciclovir treatment.

Phylogenetic Analysis of Pines Based on Chloroplast trnT-trnL Intergenic Spacer DNA Sequences

  • Um, Yurry;Park, Won-Kyu;Jo, Nam-Su;Han, Sim-Hee;Lee, Yi
    • Journal of Forest and Environmental Science
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    • v.30 no.3
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    • pp.307-313
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    • 2014
  • This study was conducted to distinguish the pines that are too similar to differentiate using conventional methods. Pinus densiflora and Pinus sylvestris have similar anatomical structure. They both have window-like pits and dentate ray tracheids, so it is not easy to distinguish the plants. We tried to find molecular markers by comparing chloroplast DNA sequences to differentiate the pines growing in Korea. We used P. densiflora, P. densiflora for. multicaulis, P. sylvestris, P. rigida, P. rigitaeda, P. koraiensis, and P. bungeana for this study. We found that the non-coding intergenic region of trnT(UGU) and trnL(UAA) genes have differences among the species. We designed a primer set to amplify the region efficiently and compared the PCR product sequences using CLC Workbench programs to find the polymorphism. We could distinguish the species using the sequences of the amplified region and the sequences were reproducible from the pines collected in Korea.

Genetic Differentiation between Sheep and Goats Based on Microsatellite DNA

  • Sun, W.;Chang, H.;Ren, Z.J.;Yang, Z.P.;Geng, R.Q.;Lu, S.X.;Du, L.;Tsunoda, K.
    • Asian-Australasian Journal of Animal Sciences
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    • v.17 no.5
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    • pp.583-587
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    • 2004
  • The 7 sheep microsatellite markersOarFCB48, OarAE101, MAF33, OarFCB11, MAF70, OarFCB304 and OarFCB128, which were located on chromosomes 2, 4, 6, 9, 17 and 19, were selected to PCR in Hu sheep, Tong sheep and their closely related species,the goat. They were studied with the amplifying result of 7 microsatellite sites of Hu Sheep, Tong Sheep and goats, the data of allele number and range of allele' size of amplifying were analyzed with ANOVA. The results showed that there were no significant differences (p<0.05) in microsatellite DNA sites among 3 populations. Concerning the conservation of microsatellites in closely related species, selecting microsatellite sites located on the chromosome where the Robertsonian fusion was caused between sheep and goat, may be used in research into genetic differentiation and evolutionary relationships between sheep and goats.

Tracing the origin of fish without hatchery information: genetic management of stock enhancement for mangrove red snapper (Lutjanus argentimaculatus) in Taiwan

  • Hsu, Te-Hua;Huang, Chang-Wen;Lin, Cheng-Hui;Lee, Hung-Tai;Pan, Chieh-Yu
    • Fisheries and Aquatic Sciences
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    • v.23 no.5
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    • pp.13.1-13.7
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    • 2020
  • Stock enhancement is considered to be a valuable approach for restoring fishery resources. Because no specific official institution in Taiwan is responsible for the production of fry, the released fry are purchased directly from the private sector. However, fishermen from the private industry have not established a genetic background, so the genetic composition for each batch of released fry is unclear. Mangrove red snapper (Lutjanus argentimaculatus), a prominent species released in Taiwan, was collected after its official release. One hundred and two field samples were compared with four batches of hatchery fry (n = 685) by using a microsatellite-based multiplex PCR assay. Four of the field samples (3.9%; 4/102) were revealed to be from a fish farm and most likely from a single batch. This study revealed that wild mangrove red snappers are genetically different from those originating from farms, and their origins can be traced through molecular markers, even without information on breeding stocks.