• BMB Reports
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• 제38권4호
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• pp.491-499
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• 2005

DNA-based molecular markers for differentiation of five penaeid shrimps (Penaeus monodon, P. semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) were developed based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Differentiation of P. monodon, P. semisulcatus and L. vannamei can be unambiguously carried out by PCR-RFLP of 16S $rDNA_{560}$ whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S $rDNA_{560}$. Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S $rDNA_{560}$ of an individual representing the most common mitotype of each species was cloned and sequenced. The new primer pair was designed and tested against the large sample sizes (312 bp product, N = 185). The amplification success was consistent across all species. PCR-RFLP of 16S $rDNA_{312}$ was as effective as that of 16S $rDNA_{560}$. Differentiation of all shrimp species were successfully carried out by SSCP analysis.

• 한국물환경학회지
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• 제22권4호
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• pp.590-598
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• 2006

In-situ Air sparging (IAS) is a groundwater remediation technique, in which organic contaminants volatilize into air form the saturated to vadose zone. This study was carried out to evaluate the effect of sludge and soil microbial community structure on air sparging of Total Petroleum Hydrocarbons (TPH) contaminated groundwater soils. In the laboratory, diesel (10,000 mg TPH/kg) contaminated saturated soil. The Air was injected in intermittent (Q=1500 mL/min, 10 minute injection and 10 minute idle) modes. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for experiment with sludge soil samples that were closely related to Agrococcus, Flavobacterium, Thermoanaerobacter, Flexibacter and Shewanella, etc, in the clone library. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil the fate of microorganisms in natural microbial community.

• 한국물환경학회지
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• 제24권1호
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• pp.19-29
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• 2008

Hot air sparging is a groundwater remediation technique, in which organic contaminants volatilized into hot air from the saturated to vadose zone. In the laboratory diesel (10,000 mg TPH/kg) was spiked in contaminated saturated aquifer soil. The hot air ($34.9{\pm}2.7^{\circ}C$) was injected in intermittent (Q=1,500 mL/min, 10 minute injection and 10 minute idle) modes. We performed microcosm tests using the groundwater samples to assess TPH reductive remediation activity. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for hot air sparging experiment with sludge soil samples that were closely related to Bacillus (149 bp, Firmicutes), Methlobacterium (149 bp, Euryarchaeotes), Pseudomonas (492 bp, ${\gamma}$-Proteobacteria), etc., in the clone library. In this study we find that TPH-water was reduced to 78.9% of the initial value in this experiment aquifer. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil fate of microorganisms in natural microbial community.

• 한국가축번식학회지
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• 제27권2호
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• pp.97-102
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• 2003

본 연구는 분만시 동복 신생자돈의 제대혈에서 추출한 genomic DNA를 PCR-RFLP 기법을 이용하여 농가수입증대를 위하여 육질이 불량한 PSS 돼지를 판별하는 방법을 개발하기 위한 기초실험으로써 그 결과를 요약하면 다음과 같다. 자돈 제대에서 혈액 genomic DNA를 추출하여 PCR에 의하여 증폭된 ryanodine receptor gene 영역의 산물은 자돈의 제대혈에서 1.8kb의 길이로 증폭되었음을 확인하였다. 제대혈에서 추출된 DNA 의 PCR 증폭 단편을 가지고 Hha I 제한효소로 digest 하여준 결과에서 PSS 돼지는 Yorkshire 종에서 출현하지 않았으나, Landrace 종과 Crossbred 종에서 각각 4.76%와 7.14%로 교잡종(LYD 또는 YLD)에서 더욱 많이 출현되었다. 이상의 결과로 볼 때 분만시 신생자돈의 제대혈을 채취하여 PSS 돼지를 조기에 선발하면 스트레스 감소는 물론 혈액채취의 간편성을 기대할 수 있을 것으로 판단된다.

• 미생물학회지
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• 제41권4호
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• pp.287-292
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• 2005

대청호에서 수화 발생이 빈번한 추소리 수역에서 2005년 3월 15일에 채취한 시료를 대상으로 유전자 분식에 의한 cyanobacteria의 다양성을 조사하였다. rpcBA-Intergenic Spacer (IGS)는 cyanobacteria에 특징적 색소인 phycocyanin을 합성하는 유전자와 유전자 사이의 부분으로, 환경시료에서 cyanobacteria의 다양성을 조사하기에 매우 유용한 기능 유전자이다. cpcBA-IGS를 이용하여 restriction fragment length polymorphism (RELP)으로 cyanobacteria의 다양성을 분석한 결과 Phomidium 속은 58 clones, Anabaena 속은 14 clones, Microcyxtis 속은 4 clones, Spirulina 속은 1 clone 그리고 uncultured cyanobacteria 2 clones가 존재하였다. 전반적으로 Phormidium 속이 우점하였으며, 여름철에 수화를 일으키는 Anabaena 속과 Microcystis 속도 많이 분포하였다. 따라서 cyanobacteria는 cpcBA-IGS와 같은 기능 유전자에 의한 종 동정 및 군집분석이 가능함을 보였다.

• Molecules and Cells
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• 제21권3호
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• pp.411-417
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• 2006

We have developed a polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) marker that can distinguish male-fertile (N) and male-sterile (S) cytoplasm in onions. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm-containing varieties with the restriction enzyme MspI revealed that N-cytoplasm plants have a functional MspI site (CCGG), whereas the S-cytoplasm plants has a substitution in that site (CTGG), and thus no MspI target. The results obtained using this PCR-RFLP marker to distinguish between cytoplasmic male sterile factors in 35 onion varieties corresponded with those using a CMS-specific sequence-characterized amplified region (SCAR) marker. Moreover, the PCR-RFLP marker can identify N- ot S-cytoplasms in DNA sample mixtures in which they are in up to a 10-fold minority, indicating that use of the marker has high diagnostic precision. We also demonstrated the usefulness of the SNP detected in the psbA gene for high-throughput discrimination of CMS factors using Real-time PCR and a TaqMan probe assay.

• Food Science and Biotechnology
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• 제17권4호
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• pp.888-891
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• 2008

Twenty-three fungal strains were isolated from meju that had originated from the Sunchang province, the famous location for making fermented soybean foods in Korea. The restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rDNA (ITS-RFLP) was applied to differentiate the isolated fungal strains. First, the ITS region by polymerase chain reaction (PCR) with specific primers was amplified and then cleaved the products with different restriction enzymes. Cleavage of the amplified fragments with the restriction enzymes AluI, HaeIII, HhaI, and TaqI revealed extensive polymorphisms. The ITS-RFLP results highly correlated with ITS sequence analysis. All of the 23 fungal strains were classified into 5 groups by ITS-RFLP analysis. Aspergillus oryzae was the major fungal strain isolated from Sunchang meju (12 out of 23), while Aspergillus fumigatus was the next most frequently isolated strain (7 out of 23). In contrast, it was found that Fusarium asiaticum, Aspergillus sydowii, and Arthrinium sp. were the minor fungal strains in meju.

• 한국유기농업학회지
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• 제27권3호
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• pp.353-363
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• 2019

To identify four cyst nematodes (Heterodera schachtii, H. trifolii, H. glycines, H. sojae) that are economically important plant-parasitic nematodes in Korea, restriction fragment length polymorphism (RFLP) by 8 endonucleases (PstI, VspI, AlwI, RsaI, MvaI, EcoRI, Eco72I, Hinf I) was performed based on sequence difference of mitochondrial DNA cytochrome c oxidase subunit I (COI) gene. As a result, species-specific DNA band patterns by RsaI endonuclease were observed in H. schachtii. The specific patterns was in H. trifolii by 3 endonucleases (VspI, AlwI, Hinf I), and was in H. glycines by Hinf I. While, H. sojae was not digested by 4 endonuclease (VspI, AlwI, RsaI, Hinf I). This study showed that four cyst nematodes could be distinguished using RFLP by 4 endonucleases (RsaI, VspI, AlwI, Hinf I) based on the sequence difference of COI gene.

• The Plant Pathology Journal
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• 제38권6호
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• pp.616-628
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• 2022

Resistance to pyraclostrobin due to a single nucleotide polymorphism at 143rd amino acid position on the cytochrome b gene has been a major source of concern in red pepper field infected by anthracnose in Korea. Therefore, this study investigated the response of 24 isolates of C. acutatum and C. gloeosporioides isolated from anthracnose infected red pepper fruits using agar dilution method and other molecular techniques such as cytochrome b gene sequencing, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and allele-specific polymerase chain reaction (PCR). The result showed that four isolates were resistant to pyraclostrobin on agar dilution method and possessed GCT (alanine) codon at 143rd amino acid position, whereas the sensitive isolates possessed GGT (glycine). Furthermore, this study illustrated the difference in the cytochrome b gene structure of C. acutatum and C. gloeosporioides. The use of cDNA in this study suggested that the primer Cacytb-P2 can amplify the cytochrome b gene of both C. acutatum and C. gloeosporioides despite the presence of various introns in the cytochrome b gene structure of C. gloeosporioides. The use of allele-specific PCR and PCR-RFLP provided clear difference between the resistant and sensitive isolates. The application of molecular technique in the evaluation of the resistance status of anthracnose pathogen in red pepper provided rapid, reliable, and accurate results that can be helpful in the early adoption of fungicide-resistant management strategies for the strobilurins in the field.

• Animal cells and systems
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• 제7권2호
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• pp.145-149
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• 2003

Adult periodontitis is a chronic inflammatory disease whose etiology is not well defined. Recent studies have shown that vitamin D receptor gene has been a candidate for the susceptibility of adult periodontitis. The purpose of this study is to investigate the frequency of Taq I restriction fragment length polymorphism (RFLP) in the vitamin D receptor gene in nan periodontically healthy controls and 28 adult periodontitis patients. Taq I RFLP in the vitamin D receptor gene was detected by PCR amplification, followed by restriction enzyme digestion and 2％ agarose gel electrophoresis. There was no significant difference in the distribution of Taq I RFLP between healthy controls and adult periodontitis group (P > 0.05). Thus, Taq I RFLP in the vitamin D receptor gene may not confer the susceptibility to adult periodontitis in Korean population. However, t allele distributions of this RFLP showed various frequencies among ethnic groups studied. Further studies in other ethnic groups will be required.