• Title/Summary/Keyword: Dot hybridization

Search Result 65, Processing Time 0.024 seconds

In situ Hybridization for the Detection and Localization of the Bitter Taste Receptor Tas2r108 in the Murine Submandibular Gland

  • Ki, Su-Young;Cho, Young-Kyung;Chung, Ki-Myung;Kim, Kyung-Nyun
    • International Journal of Oral Biology
    • /
    • v.41 no.2
    • /
    • pp.97-103
    • /
    • 2016
  • Mammals have 3 pairs of major salivary glands i.e., the parotid, submandibular, and sublingual glands. Saliva secretion of these glands is modulated by taste perception. Salivary glands are composed mainly of acinar and ductal cells. Primary saliva is secreted by acinar cells and modified during ductal flow. Recently, of the murine 35 bitter taste receptors, Tas2r108 was expressed at highest levels in the submandibular gland by qPCR. Further, Tas2r108-transfected cells respond to a range of bitter compounds, such as denatonium, quinine, colchicine, diphenidol, caffeine and dapson. The objective of the present study was to characterize the expression of Tas2r108 mRNA in acinar and/or ductal cells of the submandibular gland using in situ hybridization (ISH). Male 42-60 days old DBA2 mice were used in the study. Messenger RNAs were extracted from the submandibular gland for generating digoxigenin (DIG) labeled-cRNA probes. These probes were transcribed in anti-sense and sense orientation using T7 RNA polymerase. Dot blot hybridization was performed using DIG labeled-cRNA probes, in order to estimate integrity and optimal diluting concentration of these probes. Subsequently, ISH was performed on murine submandibular gland to detect Tas2r108 mRNA. Dot blot hybridization data demonstrated that Tas2r108 DIG labeled-cRNA anti-sense probes specifically detected Tas2r108 cDNA. ISH results showed that the anti-sense probes labeled acinar and ductal cells in the submandibular gland, whereas no staining was visible in sense controls. Interestingly, the Tas2r108 expression levels were higher in acinar than ductal cells. These results suggested that Tas2r108 might be more associated with primary saliva secretion than with ductal modification of saliva composition.

Genomic DNA probe and purification of Theileria sergenti merozoites in Korean cattle (한우에 감염된 Theileria sergenti merozoite의 순수분리와 genomic DNA probe에 관한 연구)

  • Chae, Joon-seok;Lee, Joo-mook;Kwon, Oh-deog;Chae, Keon-sang
    • Korean Journal of Veterinary Research
    • /
    • v.34 no.2
    • /
    • pp.387-394
    • /
    • 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.

  • PDF

생물 공학 의약품의 품질관리에 관한 연구(비방사능 물질 표지법을 이용한 숙주유래 DNA의 검출법 개발)

  • 용군호;민홍기;김창민;오호정;한강현;최규실
    • Proceedings of the Korean Society of Applied Pharmacology
    • /
    • 1993.04a
    • /
    • pp.59-59
    • /
    • 1993
  • 비방사능물질인 Biotin을 DNA probe에 표지하여 nonisotopic hybridization 방법을 사용하여 감도를 높임으로써, 손쉽게 생물공학 의약품의 품질관리에 사용되도록 하였다. Bethesda Research Laboratories(BRL) 회사 제품인 biotinylated probes-avidin alkaline phosphatase를 이용한 chemiluminescene detection방법으로 행하여 λ phage DNA, yeast DNA, E.coil DNA의 한계 검출 농도를 알아내고, 생물 공학 제품에 적용하였다. Dot blot hybridization 방법으로 행하여 λ phage DNA는 0.1pg, Yeast DHA는 4.5pg, E. coli DNA는 8.9pg까지 검출되었고, 기존 생물공학 제품에서는 숙주 유래 DNA가 전혀 검출되지 않았다.

  • PDF

Comparative analyses of Theileria sergenti isolated from Korea and Japan by southern hybridization and polymerase chain reaction (Sourthern hybridization과 중합효소연쇄반응을 이용한 한국과 일본의 Theileria sergenti 비교)

  • Chae, Joon-seok;Lee, Joo-mook;Kwon, Oh-deog;Lee, Seung-ok;Chae, Keon-sang;Onuma, Misao
    • Korean Journal of Veterinary Research
    • /
    • v.36 no.1
    • /
    • pp.187-193
    • /
    • 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.

  • PDF

Study on isolation of Prevotella nigrescens 9336- specific DNA probes using random cloning method (무작위 클로닝법을 이용한 Prevotella nigrescens 9336 특이 DNA 프로브의 개발에 관한 연구)

  • Gang, Soon-Won;Kim, Se-Hoon;kim, Dong- Ki;Seong, Jin-Hyo;Kim, Byung-Ock;Kim, Jung- Ki
    • Journal of Periodontal and Implant Science
    • /
    • v.32 no.2
    • /
    • pp.269-280
    • /
    • 2002
  • The purpose of this study is to develop species-specific DNA probes and polymerase chain reaction (PCR) primers for detection and identification of Prevotella nigrescens (P. nigrescens) 9336. This study procedure includes (1) whole-genomic DNA extraction of P. nigrescens 9336 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse Dot Hybridization method, (4) confirmation of strain-specific DNA probe by Southern blot analysis, (5) determination of nucleotide sequences of strain-specific DNA probe. Thirty-five restriction fragments of P. nigrescens 9336 genomic DNA digested with the Hind III were obtained. Reverse dot hybridization and Southern blot analysis data showed that three of them, Pn10, Pn23, and Pn35, could be P. nigrescens 9336-specific DNA probes. These data indicated that these DNA probes could be useful in detection and identification of the P. nigrescens 9336.

Development of Streptococcus sanguinis-, Streptococcus parasanguinis-, and Streptococcus gordonii-PCR Primers Based on the Nucleotide Sequences of Species-specific DNA Probes Screened by Inverted Dot Blot Hybridization

  • Park, Soon-Nang;Kook, Joong-Ki
    • International Journal of Oral Biology
    • /
    • v.38 no.2
    • /
    • pp.43-49
    • /
    • 2013
  • The objective of this study was to develop PCR primers that are specific for Streptococcus sanguinis, Streptococcus parasanguinis, and Streptococcus gordonii. We designed the S. sanguinis-, S. parasanguinis-, and S. gordonii-specific primers, Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 respectively, based on the nucleotide sequences of the Ssa21, Spa17, and Sgo41 DNA probes that were screened using inverted dot blot hybridization (IDBH). The species-specificity of these primers was assessed against 43 strains of mitis group streptococci, including clinical strains of S. sanguinis, S. parasanguinis, and S. gordonii. The resulting PCR data revealed that species-specific amplicons had been obtained from all strains of the target species tested, and that none of these amplicons occurred in any other strains from other species. These results suggest that the Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 primers may be useful in detecting S. sanguinis, S. parasanguinis, and S. gordonii at the species level, respectively.

Analysis of Genes Involved in the Pathogenesis of Intracellularly Survival Bacteria (세포내 기생세균의 병원성 관련 유전자의 분석에 관하여)

  • Jeon, Tae-Il;Lee, Tae-Yoon;Kim, Sung-Kwang
    • Journal of Yeungnam Medical Science
    • /
    • v.9 no.2
    • /
    • pp.248-255
    • /
    • 1992
  • Eight bacterial strains were examined whether they have phoP/phoQ genes which were known to be involved in the intracellular survival of Salmonella typhimurium. The phoP/phoQ operon were known to sense the stimuli of the genes involved in the adaptation of the environment. Using 514-basepairs EcoRV DNA fragment of phoP region of Salmonella typhimurium as a probe, dot blot hybridization were performed. Chromosomal DNAs of Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marscescens, Enterobacter cloacae, Salmonella typhimurium, Escherichia coli, Shigella dysenteriae, and Listeria monocytogenes were examined by DNA hybridization assay. Against our expectation, intracellular pathogen, L. monocytogenes, did not have similar DNA sequences to phoP/phoQ of S. typhimurium, while E. coli, S. dysenteriae, and E. cloacae showed the positive signal even though they were not intracellular pathogens. This result suggested that the phoP/PhoQ operon was absent in intracellular pathogenic bacterias other than S. typhimurium. Rather it was found in phylogenetically closer bacterias to S. typhimurium, which were not able to survive in intracellular environment. Some different mechanism, which is not dependent on phoP/PhoQ operon, could be involved in the intracelluar survival of L. monocytogenes.

  • PDF

Study on development of DNA probe for identification of Prevotella intermedia G8-9-3 (Prevotella intermedia G8-9K-3을 동정할 수 있는 DNA 프로브의 개발에 관한 연구)

  • Bak, Jong-Sung;Kim, Se-Hoon;kim, Dong-Ki;Seong, Jin-Hyo;Kim, Byung-Ock;Kim, Jung-Ki
    • Journal of Periodontal and Implant Science
    • /
    • v.32 no.2
    • /
    • pp.281-290
    • /
    • 2002
  • The purpose of this study is to develop species-specific DNA probe for detection and identification of Prevotella intermedia (P. intermedia) G8-9K-3. This study procedure includes (1) whole-genomic DNA extraction of P. intermedia G8-9K-3 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse dot hybridization, (4) confirmation of strain-specific DNA probe by Southern blot hybridization, (5) determination of nucleotide sequences of strain-specific DNA probe. Twenty-eight recombinant plasmids containing Hind III-digested DNA fragments of P. intermedia G8-9K-3 were obtained. Reverse Dot Hybridization and Southern blot analysis data showed that one of them, Pig3, could be P. intermedia G8-9K-3-specific DNA probe. This datum indicates that this Pig3 DNA probe could be useful in detection and identification of the P. intermedia G8-9K-3 strain.

gyrA Mutations Found Among Ofloxacin-resistant Mycobacterium tuberculosis is Isolated from Korea

  • Kim Junho;Kim Yeun;Bae Kiho;Song Taek-Sun;Cho Sang-Nae;Lee Hyeyoung
    • Biomedical Science Letters
    • /
    • v.11 no.4
    • /
    • pp.465-471
    • /
    • 2005
  • Ofloxacin has antimycobacterial activity that possibly contributes a pivotal role in the second-line drug regimens that are used for the treatment of multidrug-resistant tuberculosis. However, in some communities, the resistance rate of Mycobacterium tuberculosis to this agent is surging. Therefore, a rapid and accurate method that can be used to determine the resistance of M tuberculosis to the ofloxacin can be very useful for effective treatment of the patients. As an effort to develop such a method, this study was set up to reveal general types of mutations that are related to ofloxacin resistance of M tuberculosis. From previous studies, it has been well known that ofloxacin resistance is associated with mutations in a gene encoding the gyrase A subunit protein. In this study, we obtained 43 ofloxacin-resistant and 50 ofloxacin-susceptible M tuberculosis clinical isolates from Masan National TB Hospital, and sequences of DNA fragment of 320 bp, region of gyrA corresponding to the ofloxacin resistance-determining region were analyzed. In brief, the results showed that a total of seven mutation types were found at gyrA. Theses mutations were all clustered within nucleotides 2574 to 2586 of the gyrA gene (codons 88 to 94). Codon 94 was the most frequently substituted site. Twenty-four of the 43 isolates had mutations at this position resulting in a total of five different types of amino acid changes $(Asp{\to}Ala,\;Asp{\to}Gly,\;Asp{\to}His,\;Asp{\to}Tyr,\;and\;Asp{\to}Asn)$. Five isolates contained a mutation at codon 90 resulting $Ala{\to}Val$ change. Four isolates had mutations at codon 91 causing a $Ser{\to}Pro$ change at this site. Two isolates contained a mutation at codon 88 and each of them resulted in different types of amino acid changes $(Gly{\to}Cys,\;Gly{\to}Ala)$. On the other hand, polymorphic site at codon 95 was found in both ofloxacin-resistant and ofloxacin-susceptible isolates. From these results, we concluded that the rate of mutations present in gyrA among ofloxacin-resistant M. tuberculosis in Korea is similar to the general rates of mutations found throughout the world. Subsequently, an oligonucleotide probe was designed based on the results of sequence analysis and was used to develop a dot blot hybridization assay system to determine ofloxacin-resistance of M tuberculosis. To evaluate this probe, dot-blot hybridization was carried out using other 57 clinical isolates, and the results showed that the dot-blot hybridization assay is good for detecting sequence alterations atgyrA gene.

  • PDF

DNA Chip using Single Stranded Large Circular DNA: Low Background and Stronger Signal Intensity

  • Park, Jong-Gu
    • Biomedical Science Letters
    • /
    • v.10 no.2
    • /
    • pp.75-84
    • /
    • 2004
  • Massive identification of differentially expressed patterns has been used as a tool to detect genes that are involved in disease related process. We employed circular single stranded sense molecules as probe DNA for a DNA chip. The circular single stranded DNAs derived from 1,152 unigene cDNA clones were purified in a high throughput mode from the culture supernatant of bacterial transformants containing recombinant phagemids and arrayed onto silanized slide glasses. The DNA chip was examined for its utility in detection of differential expression profile by using cDNA hybridization. Hybridization of the single stranded probe DNA were performed with Cy3- or Cy5-labeled target cDNA preparations at $60^\circ$C. Dot scanning performed with the hybridized slide showed 29 up-regulated and 6 down-regulated genes in a cancerous liver tissue when compared to those of adjacent noncancerous liver tissue. These results indicate that the circular single stranded sense molecules can be employed as probe DNA of arrays in order to obtain a precious panel of differentially expressed genes.

  • PDF