• 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.

• 한국수산과학회지
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• 제50권6호
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• pp.806-811
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• 2017

A multiplex PCR system comprising 14 microsatellite markers was developed for genotyping analysis of the sea cucumber Stichopus japonicus. A total of 286 samples were used to evaluate genetic polymorphisms and forensic parameters of the microsatellite loci. In a single PCR reaction, all 14 loci were uniformly amplified and a total of 269 alleles were identified. The AJ19024 locus had the largest number of alleles (46), and its discriminatory power and exclusion power were 0.99 and 0.76, respectively. The fewest alleles (8) were present at the Psj2575 locus, which provided the lowest discriminatory power (0.81) and exclusion power (0.20). The mean number of alleles, mean heterozygosity, mean discrimination power and mean exclusion power per locus were 19.21, 0.70, 0.93, and 0.46, respectively. The combined matching probability for the 14 loci was $9.64{\times}10^{-19}$, and the combined power of exclusion was 0.999995. Thus, the forensic parameters evaluated in the present study demonstrated the utility of our multiplex PCR system for biological tracing methods, such as individual identification and paternity testing, in the sea cucumber.

• 대한수의학회지
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• 제44권4호
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• pp.533-537
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• 2004

In this study, we performed a PCR-RFLP analysis of NADH oxidase gene(nox) for the characterization of porcine intestinal spirochetes isolated from Korea by the comparison with Brachyspira hyodysenteriae and B. pilosicoli reference strains. Eleven strains including four reference strains, B. hyodysenteriae B204, B234, B169, B. pilosicoli P43/6/78 and seven Korean isolates were used. PCR products of 939 bp were amplified using nox-specific primers and digested with two restriction enzymes, Bfm I and Dpn II. In study using Bfm I, both strains showed no difference in fragmented size(197 and 741 bp). When use Dpn II, B. hyodysenteriae showed two bands(209 and 684 bp), however B. pilosicoli showed a single band of 896 bp. Our results indicate that nox-specific PCR-RFLP could be used as a typing method of Brachyspira species and as an epidemiological method for identifying spirochetes isolated from swine.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.112.1-112
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• 2003

Genetic diversity of Plasmodiophora brassicae from major chinese cabbage cultivating areas in Korea was analyzed by using PCR-RFLP and RAPD. Single spores of P brassicae isolated from galls of club root made induce lesion on chinese cabbage successfully. The PCR-RFLP and RAPD by primers PbITS, URP 3, 6 and OPA 7 revealed that single spore isolates showed various DNA polymorphisms among them unrelated geographic origins. These results indicate that P. brassicae population in Korea showed genetic difference among them. This study could be facilitate to identify genetic characteristics ofP. brassicae based on DNA polymorphisms between single spore isolates and to get basic information which can be used to advanced resistance breeding against club root of chinese cabbage.

• Molecules and Cells
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• 제42권3호
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• pp.228-236
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• 2019

CD4 T cells differentiate into $ROR{\gamma}t/IL$-17A-expressing cells in the small intestine following colonization by segmented filamentous bacteria (SFB). However, it remains unclear whether SFB-specific CD4 T cells can differentiate directly from naïve precursors, and whether their effector differentiation is solely directed towards the Th17 lineage. In this study, we used adoptive T cell transfer experiments and showed that naïve CD4 T cells can migrate to the small intestinal lamina propria (sLP) and differentiate into effector T cells that synthesize IL-17A in response to SFB colonization. Using single cell RT-PCR analysis, we showed that the progenies of SFB responding T cells are not uniform but composed of transcriptionally divergent populations including Th1, Th17 and follicular helper T cells. We further confirmed this finding using in vitro culture of SFB specific intestinal CD4 T cells in the presence of cognate antigens, which also generated heterogeneous population with similar features. Collectively, these findings indicate that a single species of intestinal bacteria can generate a divergent population of antigen-specific effector CD4 T cells, rather than it provides a cytokine milieu for the development of a particular effector T cell subset.

• 한국가축번식학회지
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• 제27권3호
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• pp.197-206
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• 2003

hFSH는 $\alpha$와 $\beta$ subunit으로 구성된 heterodimer로서 두 subunit의 조합은 활성을 지닌 호르몬의 생산에 있어서 매우 중요한 단계이다. 이 조합과정의 효율을 증대하기 위하여 본 연구에서는 hFSH를 단일사슬의 단백질로 구축하고자 하였으며, 이의 일환으로 각 subunit 대한 cDNA단편을 연결하는 서열로 CTP linker를 도입하였다. 재조합한 hFSH-CTP 유전자는 pseudotype의 retrovirus vector system을 이용하여 CHO 세포와 닭의 배로 각각 전이되었다. CHO 세포에서의 FSH의 생산은 $\alpha$와 $\beta$를 각각 전이한 경우에 비해 hFSH-CTP를 전이한 경우에서 17배 이상 높은 것으로 나타났다. 닭에서는 유전자 전이를 시도한 62개체 중에서 11마리가 부화하였으며 그 중 10마리가 형질전환된 닭인 것으로 RT-PCR을 통하여 확인되었다. 그러나 개체의 혈중 FSH의 생산은 확인하지 못하였다. 이상의 실험 결과를 바탕으로 하여 재조합된 hFSH-CTP는 FSH의 발현에 매우 효율적인 구조로 생각되며, 또한 retrovirus를 이용한 유전자 전이 방법은 형질전환 가금의 생산에 있어서 매우 적절한 방법으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제21권2호
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• pp.155-160
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• 2008

Several studies have reported quantitative trait loci (QTL) for meat quality on porcine chromosome 2 (http://www.animalgenome.org/QTLdb/pig.html). For application of the molecular genetic information to the pig industry through marker-assisted selection, single nucleotide polymorphism (SNP) markers were analyzed by comparative re-sequencing of polymerase chain reaction (PCR) products of 13 candidate genes with DNA from commercial pig breeds such as Berkshire, Yorkshire, Landrace, Duroc and Korean Native pig. A total of 34 SNPs were identified in 15 PCR products producing an average of one SNP in every 253 bp. PCR restriction fragment length polymorphism (RFLP) assays were developed for 11 SNPs and used to investigate allele frequencies in five commercial pig breeds in Korea. Eight of the SNPs appear to be fixed in at least one of the five pig breeds, which indicates that different selection among pig breeds might be applied to these SNPs. Polymorphisms detected in the PTH, CSF2 and FOLR genes were chosen to genotype a Berkshire-Yorkshire pig breed reference family for linkage and association analyses. Using linkage analysis, PTH and CSF2 loci were mapped to pig chromosome 2, while FOLR was mapped to pig chromosome 9. Association analyses between SNPs in the PTH, CSF2 and FOLR suggested that the CSF2 MboII polymorphism was significantly associated with several pork quality traits in the Berkshire and Yorkshire crossed F2 pigs. Our current findings provide useful SNP marker information to fine map QTL regions on pig chromosome 2 and to clarify the relevance of SNP and quantitative traits in commercial pig populations.

• 분석과학
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• 제17권1호
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• pp.53-59
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• 2004

현재 일반적으로 많이 사용되는 single strand conformation polymorphism (SSCP)나 denaturing gradient gel electrophoresis (DGGE)같은 돌연변이 검출법은 많은 시간과 비용, 그리고 노동력이 소모된다는 단점과 실험자의 실험에 대한 숙련도에 의해 실험 결과가 달라지는 한계점을 가지고 있다. 이런 단점들을 보완하기 위하여 ion-pair reversed phase chromatography (IP-RPC)방식을 이용한 denaturing high performance liquid chromatography (DHPLC)방법을 사용하여 기관지 천식 (bronchial asthma)을 조절하는 베타2-교감신경수용체 유전자의 돌연변이를 검출하였다. 80명의 천식 환자의 혈액에서 genomic DNA를 추출하여 중합효소연쇄반응 (polymerase chain reaction)을 이용해 증폭하고, 그 산물을 SSCP와 DHPLC로 분석하였다. 그 결과, 베타2-교감신경수용체 유전자에서 SSCP는 80명의 sample 가운데 19개 (23.75%)의 돌연변이를 검출하였고, DHPLC는 25개 (31.25%)의 변이를 검출하였다. 돌연변이 검출법으로 DHPLC 분석법이 SSCP보다 더 빠르고 효과적인 방법임을 확인하였다.

• International Journal of Industrial Entomology and Biomaterials
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• 제45권1호
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• pp.29-34
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• 2022

Silkworms have recently attracted attention as healthy functional foods. Different varieties of silkworms have functional differences; thus, there is an emerging need for variety identification. In this study, we sequenced complete mitochondrial genomes (mitogenomes) of ten government-recommended silkworm varieties (BaekHwang, BaekOk, DaeBaek, DaeBak, DaeHwang, GoldenSilk, HanSaeng, JooHwang, KumKang, and KumOk). Comparison of these sequences allowed us to select the single nucleotide polymorphisms (SNPs) in 34 sites that are specific to six silkworm varieties: 13 in DaeBak, 8 in GoldenSilk, 9 in KumKang, 2 in BaekHwang, 1 in BaekOk, and 1 in DaeHwang. Among these each one SNP per variety was amplified by preparing variety-specific primers and then using tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR). As a result, it was possible to identify these six varieties among the ten silkworm varieties, evidencing that SNPs developed from mitogenomes are useful marker for the discrimination of genetically closer silkworm varieties.

• 식물병연구
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• 제15권2호
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• pp.57-62
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• 2009

우리나라 벼에 발생하는 주요 바이러스 중 애멸구에 의하여 전염하는 벼줄무의잎마름병(RSV)과 벼검은줄오갈병(RBSDV)에 대한 간편한 유전자 진단법인 VCHT-PCR 방법을 개발하였다. 벼 잎의 경우 즙액 추출 완충액은 0.5% sodium sulfite를 첨가한 0.01 M 인산완충액(pH 7.0)을 기본 완충액으로 이용하고 애멸구를 진단할 경우에는 기본 완충액에 2% PVP을 첨가하였을 때 VC/RT-PCR 진단이 잘 되었다. VC/RT-PCR을 이용한 진단에 적합한 RSV와 RBSDV 프라이머를 선발하였고 이것을 이용하여 동시진단으로 경기도 김포, 평택, 시흥지역에서 채집한 애멸구의 RSV와 RBSDV의 보독충을 쉽고 경제적으로 진단 할 수 있었다. ELISA에 의한 진단결과와 비교할 때 세 지역을 합하여 RSV에 대한 평균 보독충률은 9.2%로 동일하였으나 보독충 중 일부가 ELISA와 VC/RT-PCR 두 방법에 의한 진단결과가 다르게 나온 것은 RSV의 혈청학적, 유전적 계통 존재 가능성을 제시하고 있다. 포장에서 채집한 애멸구의 VC/RT-PCR 진단효율은 RSV와 RBSDV를 동시에 진단하므로 써 RSV만 진단이 가능한 ELISA 결과 보다 3.2% 높았다.