• Genomics & Informatics
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• 제4권1호
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• pp.1-10
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• 2006

Epigenetic alterations are common features of human solid tumors, though global DNA methylation has been difficult to assess. Restriction Landmark Genomic Scanning (RLGS) is one of technology to examine epigenetic alterations at several thousand Notl sites of promoter regions in tumor genome. To assess sequence information for Notl sequences in RLGS gel, we cloned 1,161 unique Notl-linked clones, compromising about 60% of the spots in the soluble region of RLGS profile, and performed BLAT searches on the UCSC genome server, May 2004 Freeze. 1,023 (88%) unique sequences were matched to the CpG islands of human genome showing a large bias of RLGS toward identifying potential genes or CpG islands. The cloned Notl-loci had a high frequency (71%) of occurrence within CpG islands near the 5' ends of known genes rather than within CpG islands near the 3' ends or intragenic regions, making RLGS a potent tool for the identification of gene-associated methylation events. By mixing RLGS gels with all Notl-linked clones, we addressed 151 Notl sequences onto a standard RLGS gel and compared them with previous reports from several types of tumors. We hope our sequence information will be useful to identify novel epigenetic targets in any types of tumor genome.

• Journal of Genetic Medicine
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• 제2권1호
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• pp.17-22
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• 1998

The Lesch-Nyhan syndrome which is caused by the deficiency of hypoxanthine guanine phosphoribosyltransferase is an X-linked recessive disorder characterized by hyperuricemia, choreoathetosis, mental retardation and compulsive self-injurious behavior. Clinical management of the patients with the Lesch-Nyhan syndrome is frustrating and requires burdensome medical treatment since it cripples the patient and shortens the life span by progression of neurological symptoms, but there are no cures or measures for relieving relentless natural course of the disease yet. Therefore, prenatal diagnosis of the affected fetus is important in genetic counselling for the family at high risk. In this study, four different mutations in the HPRT gene of four probands have been identified in four unrelated families; K215X, Q109X, nt.631 ${\Delta}A$, and nt.289 ${\Delta}GT$. Two mutations among them altered restriction enzyme sites; SpeI for Q109X and MaeI for nt.289 ${\Delta}GT$. Based on their molecular defects, prenatal diagnoses of 3 the fetuses were successfully made between ninth and eleventh week of gestation by polymerase chain reaction (PCR), restriction digestion and DNA sequencing using cDNA obtained from chorionic villus samples (CVS). We predicted the outcome of all fetuses prenatally. Among the three fetuses two were male and one was female according to the identification made by PCR amplification of the sex determining region of the Y chromosome(SRY) gene. Each carried a wild type allele for the corresponding mutant allele. They were also tested postnatally for the mutations to be unaffected.

• Journal of Microbiology and Biotechnology
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• 제20권6호
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• pp.1022-1026
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• 2010

The different cleavage patterns of pYBamy59 plasmid isolated from E. coli $DH5{\alpha}$ and B. longum MG1 by the cell extract of B. longum MG1 suggested that the main reason for its low transformation efficiency was related to the restriction modification (R-M) system. To confirm the correlation between the R-M system and transformation efficiency, in vitro methylation and site-directed mutagenesis were performed in pYBamy59. Sequence analysis of pYBamy59 fragments digested by the cell extract of B. longum MG1 revealed that all fragments were generated by restriction of the sequence recognized by SacII endonuclease. When pYBamy59 from E. coli was methylated in vitro by CpG or GpC methyltransferase, it was protected from SacII digestion. Site-directed mutagenesis, which removed SacII sites from pYBamy59, or in vitro methylation of pYBamy59 showed 8- to 15-fold increases in the transformation efficiency over intact pYBamy59. Modification of the SacII-related R-M system in B. longum MG1 and in vitro methylation in pYBamy 59 can improve the transformation efficiency in this strain. The results showed that the R-M system is a factor to limit introduction of exogenous DNA, and in vitro modification is a convenient method to overcome the barrier of the R-M system for transformation.

• 한국미생물·생명공학회지
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• 제14권3호
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• pp.245-250
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• 1986

청주시 무심천의 하천수에서 항생물질에 내성을 나타내는 Gram 음성 세균을 분리하여 수질환경에서 일어나는 R 플라스미드의 전이를 연구하였다. 분리된 균주사이에서 접합에 의한 R 플라스미드의 전이는 실험실 환경에서 1.1$\times$$10^{-6}$-1.2$\times$$10^{-7}$, 하천의 수질환경에서 1.2$\times$$10^{-7}$-1.0$\times$$10^{-9}$으로 나타나, 자연의 수질환경에서도 R 플라스미드의 전이가 일어남을 확인하였다. 또 T-44 균주의 Ap$^{r}$Cm$^{r}$Tc$^{r}$ 플라스미드는 형질전환에 의하여 E. coli HB 101에 1.7$\times$$10^{-6}$의 비율로 전이되었다. 분자의 크기가 약 9.01kb로 측정된 Ap$^{r}$Cm$^{r}$Tc$^{r}$플라스미드 DNA를 제한효소로 처리한 결과 이 플라스비드에는 EcoRI과 BamHI의 절단부위가 각각 하나씩 존재하고 P-stI의 절단부위는 3개가 있었다.

• Genomics & Informatics
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• 제2권4호
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• pp.174-179
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• 2004

We developed various plasmid cloning vectors that are useful in the construction of genomic and shotgun libraries. Two medium copy vectors, pCUGlblu21 (pCb21) and pAGlblu21 (pAb21), which are resistant to kanamycin ($Km^R$) and chloramphenicol ($Cam^R$), respectively, are useful for cloning DNA inserts ranging from 5kb to 15kb. Two high copy vectors, pCUGlblu31 (pCb31) and pAGlblu31 (pAb31), containing $Km^R$ and $Cam^R$, respectively, are useful for DNA inserts less than 5kb. These vectors are well adapted for large-scale genome sequencing projects by providing choice of copy number and selectable marker. The small vector size is another advantage of these vectors. All vectors contain lacZa including multicloning sites that originated from pBluscriptllsk- for easy cloning and sequencing. Two medium copy vectors contain unique and rare cutting Swal (ATTTAAAT) restriction enzyme sites for easy determination of insert size. We developed two combined vectors, pC21A31 and pC31A21, which are combinations of (pCb21 + pAb31) and (pCb31 + pAb21), respectively. These two vectors provide four choices of vectors such as $Km^R$ and medium, $Cam^R$ and high, $Cam^R$ and medium, and $Km^R$ and high copy vectors by restriction enzyme cutting, dephosphorylation, and gel purification. These vectors were successfully applied to high throughput shotgun sequencing of rice, tomato, and brassica BAC clones. With an example of extremely biased hydro sheared 3 kb shotgun library of a tomato BAC clone, which is originated from cytogenetically defined peri-centromeric region, we suggest the utility of an additional 10 kb library for sequence assembly of the difficult-to-assemble BAC clone.

• 한국수산과학회지
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• 제28권5호
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• pp.649-658
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• 1995

붕넙치과 어류의 종간에 있어서의 유전적 분화정도를 DNA level에서 관찰하기 위하여 참가자미, 문치가자미, 돌가자미, 강가자미의 미토콘드리아 DNA를 분리하고, 6염기인식의 14제한효소로써 절단한 절단단편의 염기치환수를 계산하였다. 1) mtDNA 총염기대수는 4종 모두 17.6kbp 부근으로 나타나 동일한 염기대수를 가지는 것으로 추정되었다. 2) 14종류의 제한효소로써 절단한 절단단편의 pattern으로 4종의 종내 및 종간의 haplotype수를 조사한 결과, 참가자미에서는 10개, 문치가자미에서는 4개, 강가자미에서는 2개, 돌가자미에서는 1개의 haplotype이 관찰되었으며, 종간에 있어서는 공통적인 haplotype가 전혀 관찰되지 않았다. 3) mtDNA의 유전적 변이성을 나타내는 haplotype 다양도 (h)는 참가자미에서 0.588, 문치가자미에서 0.371로 나타나 참가자미에서 높은 변이성을 나타내었다. 4) 4종의 유전적 분화의 정도를 mtDNA haplotype간의 제한 부위당 염기치환수 (d)로써 살펴 본 결과, 종내의 평균은 0.0045, 종간의 평균은 0.0344, 속간의 평균은 0.0457로 되어 종간, 속간의 값이 종내에 비해 현저하게 높은 수치를 나타내었다. 5) 4종간의 mtDNA haplotype는 1개 또는 2개의 염기치환에 의한 차이가 아니고 유전적 불연속을 나타내었다.

• 대한수의학회지
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• 제41권2호
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• pp.157-165
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• 2001

Amplified fragment length polymorphism(AFLP) technique is based on the polymorphism detection through selective PCR amplification of restriction fragments from digested genomic DNA and thus includes the procedures of the total DNA digestion by endonucleases, ligation of adapters to the ends of the fragments, and following the selective amplification of the restricted DNA fragments. This study were aimed to : (1) determine the genetic variability of S aureus strains, (2) estimate genetic diversity within and among these strains, (3) compare phylogenetic relationships among these strains as genetic markers using AFLP techniques. Genomic DNA was digested with a particular combination of three restriction enzymes with specific recognition sites and the DNA fragments were ligated to restriction specific adapters and amplified using the selective primer combinations. In the S aureus strain, the number of scorable AFLP bands detected per each primer combination varied from 29 to 102, with an average of 61.59 using 27 primer combinations. A total of 1,663 markers were generated, 904 bands of which were polymorphic, showing a 33.48% level of polymorphism with these primer combinations. Among the primer combinations, E02/T02, E02/T03, E04/H02, E02/T01 and E04/H03 primer combinations showed a high level of polymorphism with 0.78, 0.76, 0.74, 0.71 and 0.70, respectively. But T03/H01, E01/T02 and E01/T03 primer combinations showed a low level of polymorphism with 0.38, 0.37 and 0.15, respectively, Therefore, the former primer combinations will be the most effective for AFLP analysis of S aureus. In SA1 sub-types the level of polymorphism of S aureus KCTC 1927 was similar to that of S aureus CU 01(0.825) and higher than those of other strains such as S aureus CU 02 (0.715), S aureus KCTC 2199(0.625), S aureus KCTC 1916(0.607) and S aureus KCTC 1621 (0.553). In SA2 sub-types the level of polymorphism of S aureus CU 07 was similar to that of S aureus CU 08(0.935) and higher than those of both S aureus CU 04(0.883) and S aureus CU 05(0.883) and lower than those of S aureus CU 03(0.583). In SA3 subtypes the level of polymorphism of S aureus CU 11 was similar to that of S aureus CU 12(0.913) and lower than that of S aureus CU 15(0.623). The results proved that AFLP marker analysis of S aureus strain could be used to study the epidemiology of mastitis and in addition, common genotype in geographic region could be useful for the development of an effective vaccine or DNA marker for easy diagnosis of mastitis caused by S aureus infection.

• 한국수정란이식학회지
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• 제28권4호
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• pp.355-360
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• 2013

Precise, rapid and simple methods for species identification in animals are among the most important techniques in the livestock industry and research fields including meat classification. In this study, polymerase chain reaction (PCR) based molecular identification using inter species polymorphisms were examined by PCR-restriction fragment length polymorphism (RFLP) analysis for mitochondrial DNA (mtDNA) cytochrome b (CYTB) gene sequences among four mammalian livestock animals (cattle, horse, goat and pig). The results from PCR-RFLP analysis using the AluI restriction enzyme were also provided for the species-specific band patterns among CYTB gene sequences in these four species. The AluI-digestion for CYTB genes provided interesting migration patterns differentially displayed according to each species. Cattle and horse had one AluI-recognition site at different nucleotide positions and their AluI-digested fragments showed different band patterns on the gels. Pig had two AluI-recognition sites within the amplified CYTB sequences and produced three bands on the gels. Goat had no AluI-recognition site and was located at the same position as the uncut PCR product. The results showed the species-specific band patterns on a single gel among the four livestock animal species by AluI-RFLP. In addition, the results from blind tests for the meat samples collected from providers without any records showed the identical information on the species recorded by observing their phenotypes before slaughter. The application of this PCR-RFLP method can be useful and provide rapid, simple, and clear information regarding species identification for various tissue samples originating from tested livestock species.

• Restorative Dentistry and Endodontics
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• 제20권2호
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• pp.645-654
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• 1995

Porphyromonas endodontalis is a black-pigmented anaerobic Gram negative rod which is associated with endodontal infections. It has been isolated from infected dental root canals and submucous abscesses of endodontal origin. DNA probe is an available alternative, offering the direct detection of a specific microorganism. Nucleic-acid probes can be off different types: whole different: whole-genomic, cloned or oligonucleotide probes. Wholegenomic probes are the most sensitive because the entire genome is used for possible hybridization sites. However, as genetically similar species of bacteria are likely to be present in specimences, cross-reactions need to be considered. Cloned probes are isolated sequences of DNA that do not show cross-reactivity and are produced in quantity by cloning in a plasmid vector. Cloned probes can approach the sensitivity found with whole-genomic probes while avoiding known cross-reacting species. Porphyromonas endodontalis ATCC 35406 (serotype $O_1K_1$) was selected in this experiment to develop specific cloned DNA probes. EcoR I-digested genomic DNA fragments of P. endodontalis ATCC 35406 were cloned into pUC18 plasmid vector. From the E. coli transformed with the recombinant plasmid 4 clones were selected to be tested as specific DNA probes. Restriction-digested whole-genomic DNAs prepared from P. gingivalis 38(serotype a), W50(serotype b), A7A1-28(serotype C), P. intermedia 9336(serotype b), G8-9K-3(serotype C), P. endodontalis ATCC 35406(serotype $O_1K_1$), A. a Y4(serotype b), 75(serotype a), 67(serotype c), were each seperated on agarose gel electrophoresis, blotted on nylon membranes, and were hybridized with digoxigenin-dUTP labeled probe. The results were as follows: 1. Three clones of 1.6kb(probe e), 1.6kb(probe f), and 0.9kb(probe h) in size, were obtained. These clones were identified to be a part of the genomic DNA of P. endodontalis ATCC 35406 judging from their specific hybridization to the genomic DNA fragments of their own size on Southern blot. 2. The clones of 4.9kb(probe i) was identified to be a part of the genomic DNA of P. endodontalis ATCC 35406. but not to specific for itself. It was hybridized to P. gingivalis A7A1-28, P. intermedia G89K-3.

• 한국지역지리학회지
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• 제3권1호
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• pp.63-80
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• 1997

본 연구의 목적은 쓰레기 매립장의 입지선정 기준을 정립하고, 경상북도 영양군을 사례로 GIS 기법을 이용하여 실제 쓰레기 매립장의 입지후보지를 선정한 다음, 선정된 후보지를 상호비교하여 적정입지의 요인별 특성을 제시하는 것이다. 분석에 사용된 GIS소프트웨어는 Idrisi모듈인 Multi-Criteria Evaluation(MCE)이다. 영양군 쓰레기 매립장 입지선정에 이용된 입지요인은 경사도, 단층선, 기반암과 인구밀집지역, 상수원, 하천, 범람원, 도로 그리고 휴양관광지와의 거리등 9개 요인이다. 입지요인의 표준화와 요인별 가중치를 이용한 적합도와 배제기준을 적응한 결과 쓰레기 매립장 건립 불가지역은 시가지와 그 인접지역, 산악지역, 하천, 간선도로, 휴양관광지 및 그 인접지역으로 전체면적의 85.3%에 해당하는 $695.08km^2$였다. 쓰레기 매립장 적정입지 후보지로는 수비면 신암리, 청기면 행화리와 무진리 그리고 석보면 포산리등 3개지역 총 25개 지점이 선정되었다.