• Title, Summary, Keyword: PCR analysis

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ITS-PCR Analysis for the Discrimination of Moutan Cortex (목단피의 감별을 위한 ITS-PCR 분석)

  • Lee, Jae-Woong;Kim, Young-Hwa;Ko, Byoung-Seob;Ryuk, Jin-Ah;Oh, Seung-Eun;Park, Sang-Un;Lee, Mi-Young
    • Korean Journal of Medicinal Crop Science
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    • v.18 no.1
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    • pp.40-45
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    • 2010
  • The application of PCR analysis on the herbal medicine Moutan Cortex (Paeonia suffruticosa Andrews) was evaluated by the comparison of the genetic relationship based on the DNA sequence with Paeoniae Radix (Paeonia lactiflora Pallas) following development of specific primers. Moutan Cortex and Paeoniae Radix were distinguished through the PCR analysis based on the internal transcribed spacer (ITS-PCR) from nuclear ribosomal DNA region. The 294 bp PCR products both of Moutan Cortex and Paeoniae Radix was amplified by MIF1 and MIR1. And a Moutan Cortex specific 225 bp PCR amplification product was amplified by MIF2 and MIR1 primers. The 225 bp sequence could be successfully amplified from Mortan Cortex of dried herbal preparations. PCR analysis based on ITS (ITS-PCR) may be an efficient tool for the discrimination of Moutan Cortex.

Quadruplex Genotype Analysis at HumTH01, HumTPOX, HumCSF1PO and Amelogenin Loci by FoLT-PCR (FoLT-PCR에 의한 유전자형 (HumTH01, HumTPOX, HumCSF1PO & Amelogenin) 분석)

  • Lee, Yang-Han;Lim, Si-Keun;Kang, Pil-Won;Choi, Dong-Ho;Yoon, Song-Ro;Han, Myun-Soo
    • Analytical Science and Technology
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    • v.12 no.3
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    • pp.260-264
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    • 1999
  • A simple and rapid procedure, called FoLT-PCR(Formamide Low Temperature-Polymerase Chain Reaction) was applied to amplifying DNA directly from various forensic biological evidences including human blood, saliva, hair root, or semen without any DNA preparative steps. We added washing step with non-ionic detergent, 1% Triton X-100, and used Taq DNA polymerase instead of Tth DNA polymerase to amplify 3 STR loci and gender allele simultaneouly. Optimal concentration of formamide and annealing temperature were determined empirically to 8%(v/v), and $48^{\circ}C$ respectively. We also compared this method with standard PCR.

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Feasibility Study on the Fault Tree Analysis Approach for the Management of the Faults in Running PCR Analysis (PCR 과정의 오류 관리를 위한 Fault Tree Analysis 적용에 관한 시범적 연구)

  • Lim, Ji-Su;Park, Ae-Ri;Lee, Seung-Ju;Hong, Kwang-Won
    • Applied Biological Chemistry
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    • v.50 no.4
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    • pp.245-252
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    • 2007
  • FTA (fault tree analysis), an analytical method for system failure management, was employed in the management of faults in running PCR analysis. PCR is executed through several processes, in which the process of PCR machine operation was selected for the analysis by FTA. The reason for choosing the simplest process in the PCR analysis was to adopt it as a first trial to test a feasibility of the FTA approach. First, fault events-top event, intermediate event, basic events-were identified by survey on expert knowledge of PCR. Then those events were correlated deductively to build a fault tree in hierarchical structure. The fault tree was evaluated qualitatively and quantitatively, yielding minimal cut sets, structural importance, common cause vulnerability, simulation of probability of occurrence of top event, cut set importance, item importance and sensitivity. The top event was 'errors in the step of PCR machine operation in running PCR analysis'. The major intermediate events were 'failures in instrument' and 'errors in actions in experiment'. The basic events were four events, one event and one event based on human errors, instrument failure and energy source failure, respectively. Those events were combined with Boolean logic gates-AND or OR, constructing a fault tree. In the qualitative evaluation of the tree, the basic events-'errors in preparing the reaction mixture', 'errors in setting temperature and time of PCR machine', 'failure of electrical power during running PCR machine', 'errors in selecting adequate PCR machine'-proved the most critical in the occurrence of the fault of the top event. In the quantitative evaluation, the list of the critical events were not the same as that from the qualitative evaluation. It was because the probability value of PCR machine failure, not on the list above though, increased with used time, and the probability of the events of electricity failure and defective of PCR machine were given zero due to rare likelihood of the events in general. It was concluded that this feasibility study is worth being a means to introduce the novel technique, FTA, to the management of faults in running PCR analysis.

Analysis of Microbial Communities Using Culture-dependent and Culture-independent Approaches in an Anaerobic/Aerobic SBR Reactor

  • Lu Shipeng;Park Min-Jeong;Ro Hyeon-Su;Lee Dae-Sung;Park Woo-Jun;Jeon Che-Ok
    • Journal of Microbiology
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    • v.44 no.2
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    • pp.155-161
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    • 2006
  • Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63 % (137 of 217) in the standard PCR method to about 34 % (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: ${\alpha},\;{\beta},\;{\gamma}-$ Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivation-based method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.

Genotypic Identification of Cystoisospora in Immunocompromised Patients Using Tm-Variation Analysis

  • Basyoni, Maha M.A.;Elghobary, Hany Ahmed Fouad
    • The Korean Journal of Parasitology
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    • v.55 no.6
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    • pp.601-606
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    • 2017
  • Cystoisospora is responsible for morbidity in immunocompromised patients. PCR is sensitive for diagnosing Cystoisospora; however, it needs reevaluation for differential molecular diagnosis of cystoisosporiasis. We aimed at evaluating melting curve analysis (MCA) after real-time PCR (qPCR) in diagnosis and genotyping of Cystoisospora as an alternative to conventional PCR. We included 293 diarrheic stool samples of patients attending the Department of Clinical Oncology and Nuclear Medicine of Cairo University Hospitals, Egypt. Samples were subjected to microscopy, nested PCR (nPCR), and qPCR targeting the internal transcribed spacer 2 region (ITS2) of the ribosomal RNA (r RNA) gene followed by melting temperatures ($T_ms$) analysis and comparing the results to PCR-RFLP banding patterns. Using microscopy and ITS2-nPCR, 3.1% and 5.8% of cases were Cystoisospora positive, respectively, while 10.9% were positive using qPCR. Genotyping of Cystoisospora by qPCR-MCA revealed 2 genotypes. These genotypes matched with 2 distinct melting peaks with specified $T_ms$ at $85.8^{\circ}C$ and $88.6^{\circ}C$, which indicated genetic variation among Cystoisospora isolates in Egypt. Genotype II proved to be more prevalent (65.6%). HIV-related Kaposi sarcoma and leukemic patients harbored both genotypes with a tendency to genotype II. Genotype I was more prevalent in lymphomas and mammary gland tumors while colorectal and hepatocellular tumors harbored genotype II suggesting that this genotype might be responsible for the development of cystoisosporiasis in immunocompromised patients. Direct reliable identification and differentiation of Cystoisospora species could be established using $qPCR-T_ms$ analysis which is useful for rapid detection and screening of Cystoisospora genotypes principally in high risk groups.

Development of a Monitoring System for Water-borne Bacteria by a Molecular Technique, PCR-RFLP-sequence Analysis

  • Lee, Ji-Young;Jeong, Eun-Young;Lee, Kyu-sang;Seul-Ju;Kim, Jong-Bae;Kang, Joon-Wun;Lee, Hye-Young
    • Biomedical Science Letters
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    • v.9 no.3
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    • pp.139-144
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    • 2003
  • Since water borne infection causes acute diseases and results in spread of diseases by secondary infection, the prevention is very important. Therefore, it is necessary to have a method that is rapid and effective to monitor pathogenic bacteria in drinking water. In this study, we employed a systematic method, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, to develop an effective monitoring system for possible bacterial contaminants in drinking water. For this purpose, PCR primers were derived from 992 bp region of the 16s rRNA gene that is highly conserved through the different species of prokaryotes. To test whether the PCR primers designed are indeed useful for detecting all the possible microbial contaminants in the water, the primers were used to amplify 16s rRNA regions of different microbial water-borne pathogens such as E. coli, Salmonella, Yersinia, Listeria, and Staphylococcus. As expected, all of tested microorganisms amplified expected size of PCR products indicating designed PCR primers for 16s rRNA indeed can be useful to amplify all different microbial water-borne pathogens in the water. Furthermore, to test whether these 16s rRNA based PCR primers can detect bacterial populations present in the water, water samples taken from diverse sources, such as river, tap, and sewage, were used for amplification. PCR products were for then subjected for cloning into a T-vector to generate a library containing 16s rRNA sequences from various bacteria. With cloned PCR products, RFLP analysis was done using PCR products digested with restriction enzyme such as Hae III to obtain species-specific RFLP profiles. After PCR-RFLP, the bacterial clones which showed the same RFLP profiles were regarded as the same ones, and the clones which showed distinctive RFLP profiles were subsequently subjected for sequence analysis for species identification. By this PCR-RFLP analysis, we were able to reveal diverse populations of bacteria living in water. In brief, in unsterilized natural river water, over 60 different species of bacteria were found. On the other hand, no PCR products were detected in drinking tap-water. The results from this study clearly indicate that the PCR-RFLP-sequence analysis can be a useful method for monitoring diverse, perhaps pathogenic bacteria contaminated in water in a rapid fashion.

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RT- PCR Analysis of Vitellogenin Gene Expression in Bombina orientalis (무당개구리 비텔로제닌 유전자의 발현의 RT- PCR 검출법)

  • 계명찬;이명식;강희정;정경아;안혜선
    • Korean Journal of Environmental Biology
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    • v.22 no.2
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    • pp.329-335
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    • 2004
  • To develop a biomarker for the monitoring of the contamination of estrogenic endocrine disrupters in the aquatic environment, reverse transcription -polymerase chain reaction (RT-PCR) analysis of vitellogenin (Vg) mRNA expression was optimized in Bombina orientalis, a Korean red bellied toad species. Based on partial cDNA sequences of both Vg and beta actin genes of B. orientalis, specific primers for RT-PCR of Vg and beta actin mRNAs were developed. Semiquantitative RT-PCR of the Vg mRNA in liver was optimized using a beta actin mRNA as an internal control in both sexes. In female RT-PCR using $1\;\mu{g}$ of the liver cDNA resulted in a linear increment in the PCR product of Vg from 18 to 34 cycles of amplification. In male, on the contrary, the RT- PCR product was first detected at 30 cycles of amplification and a linear increment was observed from 30 to 40 cycles of amplification, suggesting that male B. orientalis expresses minute amount of Vg mRNA which is a $2^{-12}$ equivalent of female. In conclusion, the optimized protocol for semiquantitative RT-PCR analysis of Vg mRNA level in B. orientalis male liver will be useful for the environmental monitoring the xenoestrogen contamination in the freshwater environment in Korea.

Characterization of Trichoderma spp. Associated with Green Mold of Oyster Mushroom by PCR-RFLP and Sequence Analysis of ITS Regions of rDNA

  • Park, Myung-Soo;Seo, Geon-Sik;Bae, Kyung-Sook;Yu, Seung-Hun
    • The Plant Pathology Journal
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    • v.21 no.3
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    • pp.229-236
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    • 2005
  • Molecular profIles of PCR-RFLP and sequence analysis of internal transcribed spacer (ITS) regions of rDNA were compared between morphologically distinguishable species of Trichoderma isolated from substrates of oyster mushroom in Korea, T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum, T. virens, and two unidentified species, Trichoderma sp. 1 and 2. PCR­RFLP analysis divided the Trichoderma spp. into six RFLP groups, A, B, C, D, E, and F. The RFLP groups were generally agreed with described morphological species, except that the RFLP group A containing the two unidentified species. A neighbor-joining tree based on ITS sequences well supported RFLP groups observed by RFLP analysis of ITS regions of rDNA. Additionally, the two unidentified species, Trichoderma sp. 1 and 2, which could not be distinguished by PCR­RFLP analysis, were separated in sequence analysis of ITS regions of rDNA.

Gender Determination of X and Y-Specific Alphoid Repeat Sequences by PCR (PCR에 의한 X,Y-Specific Alphoid Repeat Sequences의 분석)

  • Choi, Dong-Ho;Kang, Pil-Won;Lee, Yang-Han;Han, Myun-Soo
    • Analytical Science and Technology
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    • v.12 no.1
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    • pp.80-83
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    • 1999
  • Recently, it has been possible to the alphoid gene, which has X and Y specificity, and determine the sex from human physical evidence using PCR methods. Samples from single sources, PCR method applied to the alphoid gene results in highly sensitive and accurate results even when only 60 pg of the genomic DNA was available for sex determination. Even for samples containing DNA from more than one gender source where the female DNA was present in the amount 10 times than that of the male, sex determination was possible. Therefore, this result suggests that alphoid gene, which has X and Y specificity, could be used effectively for sex determination in case of mixed DNA samples from biological evidence.

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Analysis of Differentially Expressed Genes in Kiwifruit Actinidia chinensis var. 'Hongyang' (참다래 '홍양' 품종의 차등발현유전자 분석)

  • Bae, Kyung-Mi;Kwack, Yong-Bum;Shin, II-Sheob;Kim, Se-Hee;Kim, Jeong-Hee;Cho, Kang-Hee
    • Korean Journal of Breeding Science
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    • v.43 no.5
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    • pp.448-456
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    • 2011
  • We used suppression subtractive hybridization (SSH) combined with mirror orientation selection (MOS) method to screen differentially expressed genes from red-fleshed kiwifruit 'Hongyang'. As a result, the 288 clones were obtained by subcloning PCR product and 192 clones that showed positive clones on colony PCR analysis were selected. All the positive clones were sequenced. After comparisons with the NCBI/Genbank database using the BLAST search revealed that 30 clones showed sequence similarity to genes from other organisms; 10 clones showed significant sequence similarity to known genes. Among these clones, 3 clones (AcF21, AcF42 and AcF106) had sequence homology to 1-aminicyclopropane-carboxylic acid (ACC)-oxidase (ACO) that known to be related to fruit ripening. The expression patterns of differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qReal-time PCR) analysis. All the data from qReal-time PCR analysis coincide with the results obtained from RT-PCR analysis. Three clones were expressed at higher levels in 'Hongyang' than 'Hayward'. AcF21 was highly expressed in the other genes at 120 days after full bloom (DAFB) and 160 DAFB of 'Hongyang'.