• Journal of Life Science
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• v.9 no.4
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• pp.358-367
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• 1999

Thirty strains of methicillin resistant Staphylococcus aureus were obtained from the clinical isolates. In order to investigate the pursuit of the pathogens of nosocomial infection, these strains were studied for antibiotic sensitivity as well as its resistant pattern. Among the methods of hybridization which directly confirm the specific antibiotic resistant genes by means of the recently developed specific probe DNA, dot blot hybridization and southern blot hybridization were performed and these two methods were compared in their sensitivity and specificity. Strains that is sensitive to cephalothin to the subject of methicillin resistant Staphylococcus aureus were in 43%. Those that are sensitive to cefoperazone and cefuroxime were 26% and 23%, respectively. In case of MIC, MIC50 of cefoperazone was 8 $\mu\textrm{g}$/$m\ell$, and MIC90 was 128 $\mu\textrm{g}$/$m\ell$ to be the lowest. As the results of plasmid DNA electrophoresis, most of methicillin resistant Staphylococcus aureus strains had more than 4 plasmids. These plasmids digested by BamHI, methicillin resistant Staphylococcus aureus is distributed as 10 fragments with the size of 65 kb to 1.5 kb. Dot blot hybridization were performed to examine the existence of mecA gene to show the detection rate of 50%. Southern blot hybridization were done to see if DNA bands which amplify the activity of digoxigenium-labeled probe by PCR were actually PCR products of mecA gene and it showed the detection rate of 53%. It can be concluded that the southern blot hybridization seemed to be better in sensitivity and specificity when it is compared with the results of dot blot hybridization.

• Plant Breeding and Biotechnology
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• v.5 no.4
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• pp.269-281
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• 2017

With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment.

• Applied Biological Chemistry
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• v.42 no.1
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• pp.34-38
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• 1999

We observed the structure of phenylalanine ammonia-lyase gene (PAL) which is one of the best studied plant defense-related genes responding to pathogen infection by producing suberin, lignin, and phytoalexins. In tomato, at least 5 different genetic loci have been identified by genomic southern blot hybridization and nucleotide sequence analyses of partially cloned gene fragments (Lee et al. 1992). However, our results suggest that two other isoforms designated as PAL X1 and PAL X2 are located on the chromosome in tomato plant. Furthermore, the preliminary results obtained from southern blot hybridization analyses of subcloned fragment digested with several restriction endonuclease indicated that PAL X1 and PAL X2 clones contain at least two copies of PAL gene and partial nucleotide sequence analyses of each subcloned fragment with the same primer taken from known nucleotide sequence of PAL5 gene indicated that they are located side by side on the same chromosome.

• Journal of The Korean Society of Grassland and Forage Science
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• v.25 no.4
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• pp.267-274
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• 2005

To develop transgenic orchardgrass (Dactylis glomerata L.) resistant to high temperature, a thermptolerance gene, DgP23, was introduced into orchardgrass using Agrobacterium - mediated transformation method. PCR and Southern blot analyses using genomic DNA showed specific DNA band on agarose gel and hybridization signal on X- ray film in transgenic orchardgrass harboring the recombinant DgP23 gene, but not in the wild type and empty vector control plants. RT-PCR and Southern blot analyses using total RNA also showed specific DNA band and hybridization signal. Transgenic orchardgrass did not showed ny morphological aberration both in the green house and field cultivation. Thermotolerance of transgenic plants was not detected in laboratory test. but may detected in field test.

• Journal of Periodontal and Implant Science
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• v.21 no.2
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• pp.194.2-194.2
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• 1991

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.37-44
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• 1994

The homology among the genes coding for degradation of bipheny(BP) and 4-chlorobiphenyl(4CB) was comparatively analyzed by Southern hybridization in several BP/4CB degrading bacterial strains. As the hybridization results of their genomic DNAs with pcbABCD as the DNA probe, the group of Pseudomonas sp. DJ-12. P08 and P27 strain was separated by the group of P20 and P1242 strains. The P. pseudoalcaligenes KF707 showed the hybidization signal which was homologous to the group of DJ-12, but they had different restriction endonuclease sites. The pcbAB genes in pCUl recombinant plasmid from Pseudomonas sp. DJ-12 appeared to be homologous to pchAB genes in pKTF20 cloned from P. pseudoalcaligenes KF707, but the C genes in both strains were not homologous. The bphABC in pKTF20 showed the signals homologous to the cbp ACB in pAW6194 cloned from P. putida OU83, but homologous signal was not found botween the pcbABCD genes in pCUl and the cbpADCB genes in pAW6194 recombbinant plasmid.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.48-48
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• 2003

본 실험은 spermatogonia 단계에 발현하는 유전자를 찾기 위하여 suppression subtractive hybridization를 수행하였다. 기존에 mouse에서는 spermatogonia 특이적인 유전자들이 밝혀져 있기 때문에 pig에 특이적인 유전자를 찾기 위하여 pig 250days testis와 pig 60days testis를 재료로 하여 실험하였다. SSH를 통하여 254days testis에 특이적으로 발현되는 후보유전자를 7개 찾았고 25days testis와 60days testis 의 Northern blot을 통하여 25days에 과발현하고 60days에 발현의 양이 대폭 줄어드는 spermatogonia 유전자로 생각되는 후보유전자 2개를 선택하여 pig tissue northern blot, genomic DNA southern blot, RT-PCR 그리고 In-situ hybridization을 수행하였다. Tissue northern blot과 RT-PCR을 통하여 후보자 1번은 간과 폐, 난소, 정소에서 발현하고, 후보유전자 15번은 난소와 정소에서만 특이적으로 발현함을 알았다. DNA sequence analysis와 NCBI Blast search를 통하여 후보자 1번은 다른 종에서 밝혀진 유전자였고 후보유전자 15번은 어느 종에서도 밝혀지지 않은 새로운 유전자였다. Degenerated primer를 통하여 후보자 1번의 pig full sequence를 밝히고 NCBI에 등록하였다. 그리고 In-situ hybridization을 통하여 후보유전자득이 20일째 testis의 Leydic cell에서 많이 발현되고 adult testis에서는 발현이 감소하는 결과를 얻었다. 이것으로 보아 위의 두 후보유전자는 spermatogonia에 직접 관련된 유전자이기 보다는 spermatogonia의 발달에 영향을 주는 leydic cell 특이발현을 가진 유전자로 사료되어진다.

• Korean Journal of Veterinary Research
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• v.34 no.2
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• pp.387-394
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• 1994

To make the genomic DNA probe of Theileria sergenti, the merozoites were purified from bovine erythrocytes. The infected erythrocytes were lysed by Aeromonas hydrophila(Ah-1) hemolysin, and the parasites were isolated by ultracentrifugation on a Percoll discontinuous density gradient. For construction of a T sergenti genomic DNA library, T sergenti DNA was digested with Pstl and the fragments were ligated into the PstI site of pUC19 before transformation of Escherichia coli JM83. Out of thousands of transformants obtained by transformation of E coli JM83 with the genomic library, three plasmids were chosen. The sizes of the inserted DNAs were 2.9kb(2.4kb and 0.5kb) in pKTS1, 4.3kb in pKTS2 and 1.5kb in pKTS3, respectively. The DNA fragments used as probe KTS1(2.4kb), KTS2(4.3kb) and KTS3(1.5kb) were labeled digoxigenin-11-dUTP for the Southern hybridization. In Southern hybridization, all of the probes(KTS1, KTS2 and KTS3) reacted specifically to T sergenti DNA, but not to bovine leucocyte DNA. In order to find out the sensitivities of the digoxigenin-11-dUTP-labeled KTS1 and KTS3 as the probes, purified merozoite DNA and bovine DNA (control) were checked by dot blot hybridization with the probes. Both of the probes, KTS1 and KTS3, detected as minimum amount of 975pg of the T sergenti DNA, but not bovine DNA even to 500ng.

• Korean Journal of Veterinary Research
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• v.36 no.1
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• pp.187-193
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• 1996

The T sergenti DNA fragments used as probes of KTS1(2.4kb) and KTS3(1.5kb) were labeled with digoxigenin-11-dUTP for the Southern hybridization. T sergenti DNAs from different geographic locations(Korea; Chonbuk, Kyungbuk, Chungnam, Kangwon, Cheju island, Japan; Shintoku, Shintoku 9209, Shintoku 9201, Shintoku 9202, Shintoku 9102) which had been digested with Pst I and EcoR I were probed by the digoxigenin-11-dUTP-labeled KTS1 and KTS3. As the results, the samples from Chonbuk, Kyungbuk, Cheju island in Korea and Shintoku, Shintoku 9209, Shintoku 9201, Shintoku 9102 in Japan were positively reacted, but the others from the other locations not reacted. In the comformation test of T sergenti DNA from different geographic locations, all of the samples were positively detected by PCR amplification.

• The Korean Journal of Mycology
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• v.39 no.3
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• pp.235-238
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• 2011

Agrobacterium harboring vector pCHBs with hygromycin phosphotransferase(hph) and hepatitis B virus surface antigen(HBsAg)gene was transformed into the mycelial culture of Flammulina velutipes. In particular, mild NaOH solution was treated to the mycelia before Agrobacterium infection step. This was purposed to generate putative surface wounds in the mycelial cell walls. The results showed that hygromycin-resistant($hyg^r$) mycelia could be obtained only from NaOH-treated mycelia but not from intact mycelia. The integration of $hyg^r$ gene in fungal genome was confirmed by PCR. In addition, a single transgene integration and heterologous protein expression in F. velutipes could be verified by Southern blot hybridization and western blot analysis, respectively. This study demonstrated an efficient tool for the Agrobacterium-mediated transformation of F. velutipes mycelia.