• Title/Summary/Keyword: PCR/RFLP analysis

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Novel polymorphisms of dopa decarboxylase gene and their association with lamb quality traits in Indonesian sheep

  • Ratna Sholatia Harahap;Ronny Rachman Noor;Yuni Cahya Endrawati;Huda Shalahudin Darusman;Asep Gunawan
    • Animal Bioscience
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    • v.36 no.6
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    • pp.840-850
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    • 2023
  • Objective: This study aimed to investigate the polymorphisms of the dopa decarboxylase (DDC) gene and association analysis with lamb quality and expression quantification of the DDC gene in phenotypically divergent Indonesian sheep. Methods: The totals of 189 rams with an average body weight of 24.12 kg at 10 to 12 months were used to identify DDC gene polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Among 189 rams, several rams representing various sheep genotypes were used for an association study between genotypes and phenotypic traits with proc general linear model (GLM) analysis. In addition, the gene expression analysis of the DDC mRNA in the phenotypically divergent sheep population was analyzed using quantitative reverse-transcription PCR. Results: The DDC gene (g. 5377439 G>A) showed polymorphisms that indicated three genotypes: AA, AG, and GG. The DDC gene polymorphism was significantly associated (p≤0.05) with carcass characteristics including carcass percentage, carcass length, hot and cold carcass; physical properties of lamb quality including pH value; retail cut carcass; fatty acid composition such as fat content, pentadecanoic acid (C15:0), tricosylic acid (C23:0), lignoceric acid (C24:0), oleic acid (C18:1n9c), elaidic acid (C18:1n9t), nervonic acid (C24:1), linoleic acid (C18:2n6c), arachidonic acid (C20:4n6), cervonic acid (C22:6n3); and mineral content including potassium (K). The GG genotype of the DDC gene had the best association with lamb quality traits. The DDC gene expression analysis mRNA showed no significant difference (p≥0.05) between lamb quality traits. Conclusion: The DDC gene could be used as a potential candidate gene to improve lamb quality.

Phylogenetic Analysis of Phenanthrene-Degrading Sphingomonas

  • Han, Kyu-Dong;Jung, Yong-Tae;Son, Seung-Yeol
    • Journal of Microbiology and Biotechnology
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    • v.13 no.6
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    • pp.942-948
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    • 2003
  • Soil samples were obtained from 5 sites contaminated with polycyclic aromatic hydrocarbons (PAHs). These soil samples were cultured in using phenanthrene as a sole carbon and energy source, and 36 strains of phenanthrene-degrading bacteria were isolated from 3 sites. Most of them degraded 500 ppm of phenanthrene within 8 to 10 days, and these isolates could degrade a few other PAHs other than phenanthrene. Their genotypes were determined by restriction digests of the l6S rRNA genes [amplified ribosomal DNA restriction analysis (ARDRA)]. It was found that all the phenanthrene degrading isolates were included in 4 ARDRA types, and they showed a strict site endemism. l6S rDNAs of 12 strains selected from different sites were sequenced, and they were all confirmed as Sphingomonas strains. Their l6S rDNA sequences were compared for phylogenetic analysis; their sequence showed a similar result to ARDRA typing, thus indicating that these heterotrophic soil bacteria are not regionally mixed. In addition, it was found that the microbial diversity among sampling sites could be monitored by l6S rDNA PCR-RFLP pattern alone, which is simpler and easier to perform, without l6S rDNA sequence analysis.

Human Leukocyte Antigen(HLA) Genotypes and Thyroid Autoimmunity in Korean Patients with Type 1 Diabetes (한국인 제 1형 당뇨병 환자들의 HLA 유전자형 및 자가면역성 갑상선 질환의 병발 양상)

  • Kang, So Young;Shin, Chung Ho;Yang, Sei Won;Park, Myoung Hee;Yu, Jeesuk
    • Clinical and Experimental Pediatrics
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    • v.48 no.6
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    • pp.624-633
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    • 2005
  • Purpose : This study analyzed the expression of HLA-DR and DQ genotypes and anti-thyroid autoantibodies[anti-thyroid peroxidase(TPO) and anti-thyroglobulin(TG) antibodies] in Korean patients with type 1 diabetes(T1DM) to investigate the susceptible HLA alleles to T1DM in Korea and the prevalence of thyroid autoantibodies and their significance for the development of thyroid disorders. Methods : A total of 59 Korean patients with type 1 diabetes[26 males, median age 13.7 years(range 5.7-29.9 years), diabetes duration 7.6 years(-1.7-22.5 years)] were enrolled in this study, and 200 healthy Koreans without a family history of diabetes were selected as a normal control for the comparison of HLA genotypes. Seventeen patients with anti-TPO or anti-TG were followed [median duration 3.96 years(1 day-10.7 years)] with measurement of anti-TPO, anti-TG, $T_3$, $T_4$ or free $T_4$, TSH levels and physical examinations. HLA-DR and DQ genotyping were done by PCR-SSO, PCR-SSCP, PCR-RFLP and PCR-SSP methods. Results : HLA analysis showed higher frequencies of HLA-DRB1*0301, *090102 and DQB1*0201, *030302 alleles, DRB1*0301/*090102, *090102/*090102 and DQB1 *0201/*030302, *030302/*030302, *0201/ *0302 genotypes in T1DM patients compared to controls(Pc<0.05). Fifteen(25.4 percent) had anti-TPO antibody, 12(20.3 percent) had anti-TG, 17(28.8 percent) had either autoantibody and 10(16.9 percent) had both autoantibodies. No clinical or subclinical hypothyroidism developed during follow-up after the first detection of anti-thyroid autoantibody. There was no significant correlation between thyroid autoimmunity and gender, onset age of T1DM, and diabetes duration, respectively(P>0.05). Conclusion : We thought this unique HLA-DR, DQ allele distribution might be an important factor for the low incidence of T1DM in Korea. And a high prevalence of thyroid autoantibodies in these populations suggests examinations of thyroid antibodies should be performed regularly. Optimal age for the initial screening and the frequency of re-screening for associated thyroid autoimmune diseases in T1DM remains to be determined through prospective follow-up.

Analysis of Gene Mutation and Expression Level of Follicle Stimulating Hormone Receptor in Premature Ovarian Failure(POE) Patients (조기 난소 부전증(Premature Ovarian Failure, POF) 환자에서 난포 자극 호르몬 수용체 유전자 변이 및 발현 양상에 대한 분석)

  • 김정욱;염혜원;이형송;송견지;천강우;박용석;김계현
    • Development and Reproduction
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    • v.4 no.1
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    • pp.61-66
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    • 2000
  • This study was investigated to analyze the inactivating point mutation and expression level of follicle-stimulating hormone(FSH) receptor mRNA. In first experiment, we analyzed the point mutation. Peripheral blood was collected from each patient. To screen individuals for the C566T mutation, PCR was performed for exon 7 of the FSH receptor gene in 10 patients. No inactivating point mutation of FSH receptor gene was identified in women with premature ovarian failure. To analyze the expression level of FSH receptor, mRNA expressions were examined by RT-PCR method using specific primers for the FSH receptor. The amount of FSH receptor mRNA expressed in POF patients was lower than that in the control group. But it was not significantly different. These finding suggests that lower expression of FSH receptor in premature ovarian failure patients might be the cause of the low response to the gonadotropin during the hyperstimulation in IVF-ET cycles.

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Investigation of Single Nucleotide Polymorphisms in Porcine Chromosome 2 Quantitative Trait Loci for Meat Quality Traits

  • Do, K.T.;Ha, Y.;Mote, B.E.;Rothschild, M.F.;Choi, B.H.;Lee, S.S.;Kim, T.H.;Cho, B.W.;Kim, K.S.
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.2
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    • pp.155-160
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    • 2008
  • Several studies have reported quantitative trait loci (QTL) for meat quality on porcine chromosome 2 (http://www.animalgenome.org/QTLdb/pig.html). For application of the molecular genetic information to the pig industry through marker-assisted selection, single nucleotide polymorphism (SNP) markers were analyzed by comparative re-sequencing of polymerase chain reaction (PCR) products of 13 candidate genes with DNA from commercial pig breeds such as Berkshire, Yorkshire, Landrace, Duroc and Korean Native pig. A total of 34 SNPs were identified in 15 PCR products producing an average of one SNP in every 253 bp. PCR restriction fragment length polymorphism (RFLP) assays were developed for 11 SNPs and used to investigate allele frequencies in five commercial pig breeds in Korea. Eight of the SNPs appear to be fixed in at least one of the five pig breeds, which indicates that different selection among pig breeds might be applied to these SNPs. Polymorphisms detected in the PTH, CSF2 and FOLR genes were chosen to genotype a Berkshire-Yorkshire pig breed reference family for linkage and association analyses. Using linkage analysis, PTH and CSF2 loci were mapped to pig chromosome 2, while FOLR was mapped to pig chromosome 9. Association analyses between SNPs in the PTH, CSF2 and FOLR suggested that the CSF2 MboII polymorphism was significantly associated with several pork quality traits in the Berkshire and Yorkshire crossed F2 pigs. Our current findings provide useful SNP marker information to fine map QTL regions on pig chromosome 2 and to clarify the relevance of SNP and quantitative traits in commercial pig populations.

Analysis of Melanocortin Receptor 1 (MC1R) Genotype in Korean Brindle Cattle and Korean Cattle with Dark Muzzle (칡소와 비경흑색 한우의 Melanocortin Receptor 1 (MC1R) 유전자형 분석)

  • Lee, S.S.;Yang, B.S.;Yang, Y.H.;Gang, S.Y.;Ko, S.B.;Jeong, J.K.;Oh, W.Y.;Oh, S.J.;Kim, K.I.
    • Journal of Animal Science and Technology
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    • v.44 no.1
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    • pp.23-30
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    • 2002
  • PCR-RFLP analysis was carried out to investigate the genotype of Melanocortin receptor 1 (MC1R) gene in Korean Brindle Cattle and Korean Cattle with dark muzzle, which are coat color and muzzle pigmentation variants of Korean Cattle, respectively. Allelic variants of MC1R in cattle were analyzed by digestion with BsrFⅠ, AciⅠ. Among six genotypes, $E^D/E^D,\;E^D/E^+,\;E^D/e,\;E^+/E^+,\;E^+$/e and e/e, detected in cattle, only two genotypes, $E^+/E^+\;and\;E^+$/e, were observed in Korean Brindle Cattle, probably reflecting the necessary of $E^+$ allele for the expression of black brindle coat color. As in Korean Cattle with light muzzle, the $E^+$/e and e/e genotypes were detected in Korean Cattle with dark muzzle. The $E^+$ and e alleles frequencies in two populations of Korean Cattle with dark muzzle and with light muzzle were 0.37, 0.63 and 0,11, 0.89, respectively. Although the frequency of $E^+$ allele in Korean Cattle with dark muzzle was higher than in Korean Cattle with light muzzle, the $E^+$ allele was not completely associated with dark muzzle pigmentation. The results of this experiment indicate that the difference of MC1R genotype and frequency may be useful for fixation of coat color in Korean Cattle as well as Korean Brindle Cattle.

Identification of the Polymorphisms in IFITM2 and IFITM5 Genes and their Association with Ulcerative Colitis (IFITM2 및 IFITM5 유전자다형성의 발굴과 궤양성대장염의 감수성과의 연관성)

  • Kim, Hun-Soo;Mo, Ji-Su;Alam, Khondoker Jahengir;Park, Won-Cheol;Kim, Keun Young;Chae, Soo-Cheon
    • Journal of Life Science
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    • v.25 no.1
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    • pp.84-92
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    • 2015
  • Interferon inducible transmembrane protein (IFITM) family genes have been implicated in various cellular processes such as the homotypic cell adhesion functions of IFNs and cellular anti-proliferative activities. The present study aimed to investigate whether the polymorphisms of the IFITM2 and IFITM5 genes are associated with susceptibility to UC. We identified a total of thirteen polymorphisms (eleven SNPs and two variations) in the IFITM2 gene and twelve polymorphisms (eleven SNPs and one variation) in the IFITM5 gene, by the direct sequencing method. Genotype analysis in the IFITM2 and IFITM5 SNPs was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and Taq-Man probe analysis, and the haplotype frequencies of IFITM2 and IFITM5 SNPs for multiple loci were estimated using the expectation maximization (EM) algorithm. The genotype and allele frequencies of IFITM2 SNPs, as well as IFITM5 SNPs, in UC patients were not significantly different from those of the healthy controls. We also analyzed the combined frequencies of rs77537847 of IFITM1, rs909097 of IFITM2, and rs56069858 of IFITM5 in the UC patients and the healthy controls. Although the distribution of the major combined genotype frequency did not differ significantly between the healthy controls and the UC patients, the GGT combined frequency in the healthy controls was significantly different from that in the UC patients (P=0.002). This result suggests that the combined genotype of the IFITMs polymorphisms may be associated with a susceptibility to UC and could be a useful genetic marker for UC.

Isolation and Characterization of Acidophilic Yeasts Producing Urease from Korean Traditional $Nuruk$ (전통 누룩으로부터 호산성 Urease 생산 효모의 분리 및 특성)

  • Lee, Min-Na;Park, Heui-Dong
    • Food Science and Preservation
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    • v.19 no.2
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    • pp.308-314
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    • 2012
  • Two hundred and twenty three yeast strains were randomly isolated from Korean traditional $nuruk$. Among them, six urease producing yeast strains (designated JJA, JJB, JJ22, SHA, SHC and SH10) were selected on the Christensen urea agar plates. They showed the same pattern in the PCR-RFLP analysis of the ITS I-5.8S-ITS II region digested with $Hae$III and $HinF$1 restriction endonucleases. Its DNA sequences showed 100% (strains SHA, SHC and SH10) and 99.8% (strains JJA, JJB and JJ22) identity with those of $Issatchenkia$ $orientalis$ type strain ATCC 24210. Phylogenetic analysis resulted in that all the strains were closely related to $I.$ $orientalis$. Two representative strains, JJ22 and SH10, showing the highest urease activities were selected for further characterization. Their morphological, physiological and biochemical characteristics were also the same as $I.$ $orientalis$. Therefore, both the two strains were identified as $I.$ $orientalis$. They could grow at a wide range of temperature between $20-40^{\circ}C$ as well as pH between 2.0 and 10.0. However, a higher level urease activity were obtained at acidic pH than that at alkalic pH. The maximal level of urease activity was obtained at $30^{\circ}C$ (strain SH10) or $35^{\circ}C$ (strain JJ22) and in a liquid medium adjusted to the initial pH 5.0.

An Overview for Molecular Markers in Plants (식물에서 분자 마커의 동향)

  • Huh, Man Kyu
    • Journal of Life Science
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    • v.25 no.7
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    • pp.839-848
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    • 2015
  • A molecular marker is a molecule contained within a sample taken from an organism or other matter. The development of molecular techniques for genetic analysis has led to a great contribution to our knowledge of plant genetics and our understanding of the structure and behavior of various genomes in plants. Recently, functional molecular markers have been developed to detect the presence of major genes from the analysis of pedigreed data in absence of molecular information. DNA markers have developed into many systems based on different polymorphism-detecting techniques or methods such as RFLP, AFLP, RAPD, SSR, SNP, etc. A new class of very useful DNA markers called genic molecular markers utilizing the ever-increasing archives of gene sequence information being accumulated under the EST sequencing projects on a large number of plant species. Functional markers are derived from polymorphic sequences, and are more likely to be involved in phenotypic trait variation. Based on this conceptual framework, the marker systems discussed below are all (gene)-targeted markers, which have the potential to become functional. These markers being part of the cDNA/EST-sequences, are expected to represent the functional component of the genome i.e., gene(s), in contrast to all other random DNA based markers that are developed/generated from the anonymous genomic DNA sequences/domains irrespective of their genic content/information. Especially I sited Poczai et al’ reviews, advances in plant gene-targeted and functional markers. Their reviews may be some useful information to study molecular markers in plants.

Current status of anisakid nematode larvae infection in marine fishes caught from the coastal area of Korea between 2010 and 2012 (2010~2012년 연안에서 서식하는 해산어에서 아니사키스 유충의 감염현황)

  • Kim, Wi-Sik;Jeon, Chan-Hyeok;Kim, Jeong-Ho;Kim, Do-Hyung;Oh, Myung-Joo
    • Journal of fish pathology
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    • v.25 no.3
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    • pp.189-197
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    • 2012
  • A survey was conducted to investigate infection of anisakid nematode larvae in 243 wild marine fishes caught from the southern coastal area of Korea between 2010 and 2012. The samples comprised fishes from 9 orders, 30 families and 50 species. Total infection rate of anisakid nematode larvae was 10.7% (26/243 fish), which comprised from Yeosu, 7.4% (7/95) in 2010 and 22.7% (5/22) in 2011; from Jeju, 8.2% (5/61) in 2011; from Wando, 40.9% (9/22) in 2012. Anisakid nematode larvae were not detected in Tongyoung and Wando samples in 2011. Molecular identification of the 89 worms from 26 fish was conducted by PCR-RFLP and/or sequence analysis of internal transcribed spacer (ITS) region of ribosomal DNA. From the results, 6 kinds of anisakis species were identified: Anisakis pegreffii (infection rate: 53.9%, 48/89 worms), Hysterothylacium aduncum (38.2%, 34/89), H. fabri (3.4%, 3/89), hybird (A. simplex X A. pegreffii) (2.4%, 2/89), A. simplex (1.1%, 1/89) and Raphidascaris lophii (1.1%, 1/89). The rate of single infection was 80.8% (21/26 infected fish), while 19.2% (5/26) showed mixed infection with 2 to 3 different anisakis species.