• Title/Summary/Keyword: iPCR

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Identification of Fel ursi and Cattle and Pig Bile Juices by speciesspecific PCR and PCR-RFLP (종 특이 PCR과 PCR-RFLP를 이용한 웅담과 기타 담류의 감별 방법)

  • Kwon, Ki-Rok;Baek, Seung-Il;Choi, Suk-Ho
    • Journal of Pharmacopuncture
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    • v.12 no.1
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    • pp.13-20
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    • 2009
  • Objective : This study developed species-specific PCR and PCR-RFLP to detect the adulteration of Fel ursi products with cattle and pig bile juices. Methods : All the primers for PCR and PCR-RFLP in this study were designed based on nucleotide sequences of cytochrome b genes in the mitochondria. Results : The species-specific PCR amplified a DNA fragment of 214, 214, 295, and 167 bp from Fel ursi product, bear fur, cattle bile juice, and pig bile juice, respectively. The survey using the speciesspecific PCR indicated that some of commercial Fel ursi products were adulterated with cattle and pig bile juices. PCR-RFLP using the restriction endonucleases, HaeIII and HinfI enabled differentiation among Fel ursi product, cattle bile juice, and pig bile juice. Bear furs from two animals showed variations in PCR-RFLP patterns with HaeIII. Discussion : The detection methods of the species-specific PCR and PCR-RFLP could be useful in eliminating adulterated Fel ursi products from the market.

Genetic Stock Identification of Spotted Flounder, Verasper variegatus from Yeocheun, Korea (범가자미에 대한 유전학적 동정)

  • KIM Kyung Kil;KIM Yoon;NAM Yoon Kwan;KIM Dong Soo
    • Journal of Aquaculture
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    • v.6 no.3
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    • pp.221-233
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    • 1993
  • Cell size, DNA content, chromosome and PCR-based mitochondrial 12S rRNA gene analyses were conducted to obtain basic informations for genetic stock identification of spotted flounder (Verasper variegatus) from Yeocheun, Korea. The mean erythrocytic and nuclear volumes of spotted flounder were $211.10{\mu}m^3$ and $23.03{\mu}m^3$, respectively. The haploid DNA content of this species was 0.79 pg/cell which correspond to $46.5\%$ of carp and to $22.6\%$ of mammals. Spotted flounder had the 2n = 46 acrocentric chromosomes but no heteromorphic sex chromosomes was found. Mitochondrial DNA gene for 12S ribosomal RNA was amplified by polymerase chain reaction (PCR) and the PCR products were subjected to digestion with 15 restriction endonucleases. Restriction enzyme analyses revealed that Ava I, Mae II, Sma I and Xba I had one restriction site in the mitochondrial 12S rRNA gene segment of spotted flounder, while Mae I had two. Segments of 12S rRNA gene from mitochondria in spotted flounder were sequenced and compared with channel catfish and human as controls. The 12S rRNA gene of this species was more similar to that of channel catfish than to human's.

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A Study on the Genetic Variations of Tricholoma matsutake Collected from Eleven Sites of Korea Using I-SSR PCR (I-SSR PCR을 이용한 한국의 11개 주요 산지에서 채집한 송이의 유전변이에 관한 연구)

  • Cho, Duck-Hyun;Lee, Kyung-Joon;Han, Sim-Hee
    • The Korean Journal of Mycology
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    • v.28 no.1
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    • pp.32-37
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    • 2000
  • The objectives of this study was to identify genetic variations of Tricholoma matsutake (S. Ito et Imai) Sing. growing in different geographic ranges in South Korea. Mushrooms were collected during fruiting seasons from 1994 to 1997 from 11 major sites which included four sites (Bonghwa, UIjin, Goryoung, and Chungdo) in Kyongbuk Province, three sites (Changnyung, Hadong, and Hamyang) in Kyongnam Province, two sites (Yangyang and Inje) in Kangwon Province, one site (Goisan) in Choongbuk Province, and one site (Namwon) in Chonbuk Province. Two mushrooms each from three to eight shiros in each sites were collected. Genetic characteristics were analyzed by Inter-Simple Sequence Repeat Polymerase Chain Reaction (I-SSR PCR) method using six primers. With a total of 131 DNA bands identified, Nei's genetic distance and UPGMA tree were constructed. It was estimated that genetic variations between sites amounted to 12.9%, while 87.1% of total variation was explained by variations among individuals within sites. The cluster analysis indicated that the eleven major sites were clustered into four groups, group I (Yangyang, Hamyang, Inje, Hadong and UIjin), group II (Changnyung, Namwon and Chungdo), group III (Goryoung), and group IV (Bonghwa and Goisan). It is concluded that matsutake mushrooms in South Korea have a considerable degree of genetic variations between major sites.

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Phylogenetic Analysis by RFLP and Sequencing of Mitochondrial DNA in a Korean Population

  • Lee, Jin-Young;Kim, Heui-Soo;Ha, Bae-Jin;Park, Yeong-Hong
    • Archives of Pharmacal Research
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    • v.29 no.1
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    • pp.88-95
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    • 2006
  • Analysis of molecular nature of mitochondrial DNA (mtDNA) could be powerful marker for anthropological studies of modern populations. While population genetic studies on mtDNA have been reported for several ethnic groups, no such study has been documented for the Korean population. We surveyed mtDNA polymorphisms in the HVS I of noncoding D-loop region and its upstream region from 430 unrelated healthy Korean population by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing analysis. PCR product with 2,790 bp spanning the specific mtDNA region (mt13715-16504) was subjected to RFLP analysis using 6 restriction enzyme (Hinf I, Hae III, Alu I, Dde I, Mbo I, Rsa I). On the PAUP analysis of PCR-RFLP results, 38 mtDNA haplotypes (Hap 1-38) were detected in the Korean populations, which were classified into 11 haplogroups (Grp 1-11) of related haplotypes encompassing all 38 haplotypes. In comparison of sequencing data with Anderson's reference sequence, the transition type was more prevalent than the transversion type. Insertions or deletions were not found. In addition, three of the polymorphic sites (A16240C, A16351G, G16384A) in HVS-I region are determined newly. The polymorphic sites were distributed randomly in the region, though the frequency at each site was variable. Thus, this research might be required for the genealogical study of Orientals.

Cloning and Expression of D-amino Acid Oxidise from Trigonopsis variabilis for Cephalosporin C Biotransformation (Cephalosporin C의 생변환을 위한 Trigonopsis variabilis의 D-amino Acid Oxidase 유전자의 클로닝 및 발현)

  • 이진형;정태완
    • KSBB Journal
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    • v.10 no.3
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    • pp.264-270
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    • 1995
  • Trigonopsis variabilis is a strong producer of D-amino acid oxidase that can transform cephalosporin C(ceph C) to ${\alpha}$-keto-adipyl-7-aminocephalosporanic acid(AKA-7ACA). Polymerase chain reaction (PCR) was applied to isolate the D-AAO gene from T. variabilis. To clone the PCR fragment, four different methods were examined using enzymatic reactions of Taq DNA polymerase, Klenow, T4 DNA polymerase I, Alkaline phosphatase Calf Intestinal, and T4 kinase. Ligation of phosphorylated blunt-end PCR fragment and dephosphorylated blunt-end of pUC18 plasmid yielded the best cloning efficiency One of recombinant E. coli transformants showed D-AAO activity against ceph C in both cell extracts and permeabilized cells.

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Identification of Mycobacterium species by rpoB Gene PCR-RFLP (rpoB 유전자의 PCR-RFLP를 이용한 Mycobacterium 균종 동정의 유용성)

  • Yu, Kyong-Nae;Park, Chung-Ho
    • Korean Journal of Clinical Laboratory Science
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    • v.38 no.3
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    • pp.158-165
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    • 2006
  • Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. However, Mycobacterium species has a long period of incubation, and requires serious biochemical tests such as niacin, catalase, and nitrate test that are often tedious. The development of rapid and accurate diagnostics can aid in the early diagnosis of disease caused by Mycobacterium. The current DNA amplification and hybridization methods that have been developed target several genes for the detection of mycobacterial species such as hps65, 16S rDNA, rpoB, and dnaj. These methods produce rapid and accurate results. In this study, PCR-restriction fragment length polymorphism analysis(PCR-RFLP) based on the region of the rpoB gene was used to verify the identification of non-tuburculosis Mycobacterium species. A total of 8 mycobacterial reference strains and 13 clinical isolates were digested with restriction enzymes such as Msp I in this study. The results of using this process clearly demonstrated that all 13 specimens were identified by rpoB gene PRA method. The PCR-RFLP method based on the rpoB gene is a simple, rapid, and accurate test for the identification of Mycobacterium.

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Identification of eleven species of the Pleuronectidae family using DNA-based techniques

  • Eun-Mi Kim;Mi Nan Lee;Chun-Mae Dong;Eun Soo Noh;Young-Ok Kim
    • Fisheries and Aquatic Sciences
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    • v.26 no.11
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    • pp.678-688
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    • 2023
  • Flatfish are one of the largest families in the order Pleuronectiformes and are economically important edible marine fish species. However, they have similar morphological characteristics leading to challenges in classifying correctly, which may result in mislabeling and illegal sales, such as fraudulent labeling of processed food. Therefore, accurate identification is important to ensure the quality and safety of domestic markets in Korea. Species-specific primers were prepared from the mainly consumed eleven species of the order Pleuronectiformes. To rapidly identify the 11 flatfish species, a highly efficient, rapid, multiplex polymerase chain reaction (PCR) with species-specific primers was developed. Species-specific primer sets were designed for the mitochondrial DNA cytochrome c oxidase subunit I gene. Species-specific multiplex PCR (MSS-PCR) either specifically amplified a PCR product of a unique size or failed. This MSS-PCR analysis is easy to perform and yields reliable results in less time than the previous Sanger sequencing methods. This technique could be a powerful tool for the identification of the 11 species b the family Pleuronectidae and can contribute to the prevention of falsified labeling and protection of consumer rights.

Estimation of Glomerular Filtration Rate from Plasma Creatinine and Height in Children (소아에서 신장과 혈장 Creatinine 농도를 이용한 사구체여관율 측정)

  • Kim, Jeong-Lan;Park, Yong-Hoon;Hah, Jeong-Ok
    • Journal of Yeungnam Medical Science
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    • v.5 no.1
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    • pp.93-100
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    • 1988
  • In clinical practice, creatinine clearance(Ccr) remains the most commonly used laboratory assessment of glomerular function despite methodological and technical problems of urine collection. Schwartz et al. in 1976, reported that an accurate estimate of glomerular filtration rate(GFR) could be obtained from the simple determinations of plasma creatinine(Pcr) and body length(L) : GFR($m{\ell}/min/1.73m^2$=k L(cm)/Pcr(mg/$100m{\ell}$), (k=constant). The subject of this study were ill children admitted to our pediatric department from July, 1985 to June, 1987 and they were divided into three groups; group I, from 1 to 5 years old, group II, from 6 to 10 years old, group III. from 11 to 15 years old. The results were as following ; 1) Measured creatinine clearance($Ccr_M$, $m{\ell}/min/1.73m^2$) were $109.73{\pm}9.97$ in group I, $108.26{\pm}9.02$ in group II, $96.20{\pm}4.72$ in group III and $105.48{\pm}5.23$ in all age group. 2) Measured k($k_M$) obtained from $Ccr_M=k$ Ht/Pcr were $0.49{\pm}0.03$ in group I, $0.48{\pm}0.02$ in group II, $0.43{\pm}0.02$ in group III, and $0.47{\pm}0.02$ in all age group.(Ht ; height) 3) Linear equations and correlation coefficients between Ht/Pcr(x) and Ccr(y) were y=0.822x-65.63(r=0.99) in group I, y=0.61x-23.46(r=0.72) in group II, y=0.18x+54.44(r=0.54) in group III and y=0.58x-22.13(r=0.81) in all age group. 4) $Ccr_E$ was again estiamted from linear equations between Ht/Pcr and $Ccr_M$ and $k_E$ was calculated with Ht/Pcr and $Ccr_E$ were $0.48{\pm}0.01$ in group I, $0.49{\pm}0.01$in group II, $0.43{\pm}0.01$ in group III and $0.47{\pm}0.00$ in all age group. 5) Consistant values of $k_E$ and $k_M$ were highly significant as 95~97.5% in group I and II, 90~95% in group III and 97.5~99% in all age group. In summary, we could estimate GFR with height, plasma creatinine and measured k($k_M$) according to the age in easy and rapid way.

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Rapid Methods to Distinguish Heterodera schachtii from Heterodera glycines Using PCR Technique (PCR 기법을 이용한 사탕무씨스트선충과 콩씨스트선충의 간이동정)

  • Ko, Hyoung Rai;Kim, Eun Hwa;Kim, Se Jong;Lee, Jae Kook;Lee, Wang Hyu
    • Research in Plant Disease
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    • v.23 no.3
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    • pp.241-248
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    • 2017
  • The purpose of this study was to develop rapid methods for distinguishing between Heterodera schachtii and H. glycines detected from chinese cabbage fields of highland in Gangwon, Korea. To do this, we performed PCR-RFLP and PCR with the primers set developed in this study for GC147, GC408 and PM001 population, H. schachtii, and YS224, DA142 and BC115 population, H. glycines. Eight restriction enzymes generated RFLP profiles of mtDNA COI region for populations of H. schachtii and H. glycines, repectively. As a result, treatment of two restriction enzymes, RsaI and HinfI, were allowed to distinguish H. schachtii from H. glycines based on the differences of DNA band patterns. The primer set, #JBS1, #JBG1 and #JB3R, amplified specific fragments with 277 and 339 bp of H. schachtii, 339 bp of H. glycines, respectively, while it did not amplify fragments from three root-knot nematodes and two root-lesion nematodes. Thus, the primer set developed in this study could be a good method, which is used to distinguish between H. schachtii and H. glycines.