• Title/Summary/Keyword: PCR application

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Utilization of Genetic Polymorphisms in Cattle (소에서의 유전적 다형의 이용)

  • 신형두
    • Journal of Embryo Transfer
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    • v.10 no.1
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    • pp.23-31
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    • 1995
  • Several kinds of analytic methods for genetic polymorphism in cattle, including bovine blood typing, PCR-RFLP, BoLA and microsatellite typing were described. A few respect to consider for choosing method for actual application of genetic polymorphism were emphasized. The probability of relationship between characteristics and gene concerned, repetibility and easiness of methods applied and the possibility of clarification for segregation pattern should be deliberated.

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Association of Genetic Polymorphisms of Estrogen Receptor with Litter Size using PCR-RFLP in Yorkshire Swine (Yorkshire종 돼지에서 PCR-RFLP을 이용한 Estrogen Receptor의 유전적 다형과 산자수간의 관련성)

  • Kim, J.E.;Song, W.C.;Choi, B.D.;Kho, Y.;Park, S.S.;Hong, K.C.
    • Journal of Animal Science and Technology
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    • v.45 no.4
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    • pp.523-528
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    • 2003
  • This study was performed to investigate a possible association of the porcine estrogen receptor(ER) locus with the total number of born(TNB) and number of born alive(NBA) in Yorkshire pigs. Using DNAs extracted from 242 Yorkshire pigs, the ER genotype was determined by PvuII PCR-RFLP. The ER allele frequencies of two types of A and B were 0.39 and 0.61, respectively. The least squares means of the litter size by ER genotype was evaluated. The TNB and NBA were found to be associated with an specific ER allele. The genotype at the porcine ER locus has an application potential for marker-assisted selection for litter size in pigs.

Genetic Variation in the ABCB1 Gene May Lead to mRNA Level Chabge: Application to Gastric Cancer Cases

  • Mansoori, Maryam;Golalipour, Masoud;Alizadeh, Shahriar;Jahangirerad, Ataollah;Khandozi, Seyed Reza;Fakharai, Habibollah;Shahbazi, Majid
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.18
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    • pp.8467-8471
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    • 2016
  • Background: One of the major mechanisms for drug resistance is associated with altered anticancer drug transport, mediated by the human-adenosine triphosphate binding cassette (ABC) transporter superfamily proteins. The overexpression of adenosine triphosphate binding cassette, sub-family B, member 1 (ABCB1) by multidrug-resistant cancer cells is a serious impediment to chemotherapy. In our study we have studied the possibility that structural single-nucleotide polymorphisms (SNP) are the mechanism of ABCB1 overexpression. Materials and Methods: A total of 101 gastric cancer multidrug resistant cases and 100 controls were genotyped with sequence-specific primed PCR (SSP-PCR). Gene expression was evaluated for 70 multidrug resistant cases and 54 controls by real time PCR. The correlation between the two groups was based on secondary structures of RNA predicted by bioinformatics tool. Results: The results of genotyping showed that among 3 studied SNPs, rs28381943 and rs2032586 had significant differences between patient and control groups but there were no differences in the two groups for C3435T. The results of real time PCR showed over-expression of ABCB1 when we compared our data with each of the genotypes in average mode. Prediction of secondary structures in the existence of 2 related SNPs (rs28381943 and rs2032586) showed that the amount of ${\Delta}G$ for original mRNA is higher than the amount of ${\Delta}G$ for the two mentioned SNPs. Conclusions: We have observed that 2 of our studied SNPs (rs283821943 and rs2032586) may elevate the expression of ABCB1 gene, through increase in mRNA stability, while this was not the case for C3435T.

Analyses of Dystrophin Gene and Sex Determination using PEP-PCR in Single Fetal Cells (단일 태아세포에서의 PEP-PCR을 이용한 성의 결정과 Dystrophin 유전자 분석)

  • Choi, Soo-Kyung;Kim, Jin-Woo;Cho, Eun-Hee;Park, So-Yeon;Ryu, Hyun-Mee;Kang, Inn-Soo
    • Clinical and Experimental Reproductive Medicine
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    • v.24 no.1
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    • pp.51-56
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    • 1997
  • Recently, through the development of the primer extension preamplification(PEP) method which amplifies the whole genome, simultaneous multiple DNA analysis has become possible. Whole genome from each single cell can be amplified using 15 base oligonucleotide random primer. The greatest advantage of PEP-PCR is the ability to investigate several loci simultaneously and confirm results by analysing multiple aliquots for each locus. This technique led to the development of preimplantation genetic disease diagnosis using blastomere from early embryo, sperm, polar body and oocyte. In this study, we applied PEP-PCR in 20 cases of single amniocyte and 20 cases of single chorionic villus cell for the clinical application of the prenatal and preimplantational genetic diagnosis. We analysed 7 gene loci simultaneously which are 46, 47 exons related to dystrophin gene, two VNTR (variable number tandem repeat) markers using 5'dysIII, 3'CA related to dystrophin gene and DYZ1, DYZ3, DYS14 regions on chromosome Y. In all the tests, 97.5% of PEP-PCR amplifications with single cells were successful. We obtained 38/40 (95%) accuracy in gender determination through chromosome analysis comparison. Therefore, these results have significant implications for a sperm or oocyte analysis and prenatal or preimplantational genetic diagnosis.

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Application of Molecular Methods for the Identification of Acetic Acid Bacteria Isolated from Blueberries and Citrus Fruits

  • Gerard, Liliana Mabel;Davies, Cristina Veronica;Solda, Carina Alejandra;Corrado, Maria Belen;Fernandez, Maria Veronica
    • Microbiology and Biotechnology Letters
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    • v.48 no.2
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    • pp.193-204
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    • 2020
  • Sixteen acetic acid bacteria (AAB) were isolated from blueberries and citric fruits of the Salto Grande region (Concordia, Entre Rios, Argentina) using enrichment techniques and plate isolation. Enrichment broths containing ethanol and acetic acid enabled maximum AAB recovery, since these components promote their growth. Biochemical tests allowed classification of the bacteria at genus level. PCR-RFLP of the 16S rRNA and PCR-RFLP of the 16S-23S rRNA intergenic spacer allowed further classification at the species level; this required treatment of the amplified products of 16S and 16S-23S ITS ribosomal genes with the following restriction enzymes: AluI, RsaI, HaeIII, MspI, TaqI, CfoI, and Tru9I. C7, C8, A80, A160, and A180 isolates were identified as Gluconobacter frateurii; C1, C2, C3, C4, C5, C6, A70, and A210 isolates as Acetobacter pasteurianus; A50 and A140 isolates as Acetobacter tropicalis; and C9 isolate as Acetobacter syzygii. The bacteria identified by 16S rRNA PCR-RFLP were validated by 16S-23S PCR-RFLP; however, the C1 isolate showed different restriction patterns during identification and validation. Partial sequencing of the 16S gene resolved the discrepancy.

Application of Rapid and Reliable Detection of Cymbidium Mosaic Virus by Reverse Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Immunoassay

  • Do-Hyun, Kim;Rae-Dong, Jeong;Sena, Choi;Ho-Jong, Ju;Ju-Yeon, Yoon
    • The Plant Pathology Journal
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    • v.38 no.6
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    • pp.665-672
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    • 2022
  • Cymbidium mosaic virus (CymMV) is one of economically important viruses that cause significant losses of orchids in the world. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow immunostrip (LFI) assay was developed for the detection of CymMV in orchid plants. A pair of primers containing fluorescent probes at each terminus that amplifies highly specifically a part of the coat protein gene of CymMV was determined for RT-RPA assay. The RT-RPA assay involved incubation at an isothermal temperature (39℃) and could be performed rapidly within 30 min. In addition, no cross-reactivity was observed to occur with odontoglossum ringspot virus and cymbidium chlorotic mosaic virus. The RT-RPA with LFI assay (RT-RPA-LFI) for CymMV showed 100 times more sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the RT-PCR-LFI assay demonstrated the simplicity and the rapidity of CymMV detection since the assay did not require any equipment, by comparing results with those of conventional RT-PCR. On-site application of the RT-RPA-LFI assay was validated for the detection of CymMV in field-collected orchids, indicating a simple, rapid, sensitive, and reliable method for detecting CymMV in orchids.