• 한국산림과학회지
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• 제102권2호
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• pp.223-228
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• 2013

파이토플라스마 증폭 프라이머, P1/P7 및 R16F2n/R2를 이용하여 뽕나무 품종 42개체에 대하여 뽕나무오갈병 파이토플라스마의 전염여부를 조사한 결과 공시 모두에서 파이토플라스마가 검출되었다. 뽕나무오갈병 파이토플라스마 단일염기변이를 SSCP분석기법을 응용하여 분석조건을 조사한 결과 P1/P7(약 1.8 kb) 및 R16F2n/R2(약 1.2kb)로 증폭한 PCR산물에서는 6% polyacrylamide gel 농도, 150V, $10^{\circ}C$의 전기영동 조건에서 SSCP밴드패턴이 나타났다. 유사한 SSCP밴드 패턴을 보이는 두 시료간의 밴드형태를 뚜렷하게 구별하는 방법을 찾기 위하여 뽕나무 오갈병파이토플라스마와 대추나무 빗자루병 파이토플라스마의 P1/P7 및 R16F2n/R2 프라이머로 증폭한 PCR산물을 혼합한 후 SSCP분석 결과, 전기영동상에서 대추나무 파이토플라스마와 뽕나무 파이토플라스마의 SSCP 밴드패턴 모두를 관찰할 수 있었다. 본 연구 결과, 기존에 약 600 bp 크기로 한정된 것으로 알려진 SSCP 분석법을 응용하여 파이토플라스마 PCR 산물 1.8 kb 또는 1.2 kb 크기에서도 유사한 SSCP 밴드패턴에 의하여 단일염기변이를 검출할 수 있었다.

• 한국산림과학회지
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• 제95권6호
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• pp.631-635
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• 2006

파이토플라스마 증폭 primer, R16F2n/R2를 이용하여 뽕나무 오갈병 파이토플라스마와 대추나무 빗자루병 파이토플라스마에 대하여 SSCP분석법을 이용하여 염기변이 분석을 하였다. 그 결과 뽕나무 및 대추나무 파이토플라스마는 약 1.2 kb PCR 산물을 이용하더라도 뚜렷한 밴드차이를 나타내었다. 유사한 SSCP밴드 패턴을 보이는 두 시료 간의 밴드형태를 뚜렷하게 구별하는 방법을 찾기 위하여 뽕나무 오갈병 파이토플라스마와 대추나무 빗자루병 파이토플라스마의 PCR산물을 혼합한 후 SSCP분석 결과, 전기영동상에서 대추나무 파이토플라스마와 뽕나무 파이토플라스마의 SSCP 밴드패턴 모두를 관찰할 수 있었다. 본 연구 결과, 기존에 약 600bp 크기로 한정된 것으로 알려진 SSCP 분석을 PCR 산물 1.2 kb을 이용하여 유사한 SSCP 밴드패턴을 보이는 두 시료간의 밴드형태를 두 시료의 PCR 산물을 혼합하여 SSCP분석함으로써 뚜렷하게 구별할 수 있었다.

• 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.

• 식물병연구
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• 제17권2호
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• pp.169-176
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• 2011

우리나라에서 분리한 P. katsurae의 유전적 특성을 구명하기 위하여 국내에서 분리한 P. katsurae를 대상으로 nuclear DNA(nDNA)의 ${\beta}$-tubulin (BTU)과 Elongation facter 1 alpha (EF1A) 그리고 rDNA ITS 부위의 PCR-SSCP 분석을 실시하여, P. katsurae와 Phytophthora 속에 속하는 다양한 종들의 각 부위를 대상으로 유전적 유연관계를 비교분석 하고 동정에 이용하고자 하였다. 각각의 Phytophthora 속에서 변이가 가장 많이 발생하는 부위를 포함하여 증폭 시킬 수 있도록 각 부위의 공통 염기배열로부터 제작된 primer는 Phytophthora 종에 특이적인 반응을 나타냄으로서 동정 및 진단에도 유용하게 활용될 수 있을 것으로 판단되었다. SSCP 분석 결과는 국내 P. katsurae 균주와 공시한 다른 Phytophthora 속 균주들과의 구분이 가능하였으며, Phytophthora 종 간의 구분도 가능하였다. 그러나 한 가지 부위만을 이용한 PCR-SSCP 분석은 Phytophthora 종 간의 구분이 어려운 경우도 있었다. 따라서 보다 정확하고 명확한 Phytophthora 종의 유전적 다양성 분석 및 동정을 위하여서는 단일 부위에 의한 PCR-SSCP보다는 복수 부위에 의한 PCR-SSCP를 실시하는 것이 바람직한 것으로 확인되었다.

• Journal of Microbiology
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• 제38권1호
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• pp.11-17
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• 2000

A new method was developed for the rapid analysis of diverse bacterial species in the natural environment. Our method is based on PCR-single-strands-conformation polymorphism (PCR-SSCP) and selective isolation technique of single-stranded DNA. Variable V3 fragments of 16S rDNA were amplified by PCR with bacterial 16S rDNA primers, where one of the primers was biotinylated at the 5'-end. The biotinylated strands of the PCR products were selectively isolated by using streptavidin paramagnetic particles and a magnetic stand, to prevent SSCP analysis producing heteroduplexes from heterogeneous DNA samples. The selected strands were separated by electrophoresis on a polyacrylamide gel, and detected by silver staining. Analysis of PCR products from 8 bacterial strains demonstrated their characteristic DNA band patterns. In addition, changes in the structure of the bacterial community and species diversity in the microcosm treated with phenol could be monitored. After 3 weeks of incubation, phenol and its intermediate, 2-hydroxy-muconic-semialdehyde, were degraded by indigenous bacteria. These dominating bacterial populations were identified as strong bands on an SSCP gel. Therefore, this study provides useful tools for microbial community analysis of natural habitats.

• 한국약용작물학회지
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• 제18권4호
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• pp.266-272
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• 2010

There is a considerable difference in morphological traits between Bokbunja cultivated in Korea (KCB) and Korea native Rubus coreanus, contrary to the conviction that the cultivated Bokbunja is the domestication of R. coreanus. To infer the phylogenetic relationship of KCB with other Rubus species, we compared the chloroplast DNA spacers of KCB with those of several Rubus species including black raspberry, R. occidentalis. The three chloroplast DNA spacers, atpB~rbcL, trnL~trnF, and trnT~trnL, were amplified using the specific primer pairs and converted to Single Strand Conformational Polymorphism (SSCP) markers. The SSCP makers of the chloroplast DNA spacers showed a considerable variation both within and among Rubus species. In the phylogenetic tree generated by the SSCP markers, KCB accessions were located in the same clade with R. occidentalis, but R. coreanus accessions in the different clade. Also, in the phylogenetic tree by the nucleotide sequences of the chloroplast DNA spacer trnL~trnF, KCB located in the same clade with R. occidentalis but not with R. coreanus. These results suggest that the three KCB accessions share higher similarity with R. occidentalis than with R. coreanus in the three chloroplast DNA spacers.

• Journal of Animal Science and Technology
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• 제45권6호
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• pp.911-916
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• 2003

돼지 Duroc 품종의 mitochondria DNA D-loop전체 유전자를 증폭하기 위하여 많은 동물에서 고도로 상동성이 높은 tRNA-Pro와 tRNA-Phe 염기서열 일부를 이용하여 oligonucleotide primer를 제작하였다. 그 결과 Duroc 품종의 D-loop 전체 유전자는 1,145 base pairs 였으며, 그 중간위치에 10bp의 Sus Scrofa-specific sequence (TACACGTGCG)가 10개 존재하고 있었다. 돌연변이 검출을 위하여 가장 변이가 심한 지역을 primer 제작하여 345 bp의 DNA 단편을 증폭하였으며, Single Stranded Conformation Polymorphism(SSCP) 분석은 8% polyacrylamide gel에서 200 V, 16시간 전기영동하여 ethidium bromide (EtBr)로 10분간 염색하여 UV image analyzer로 관찰하였다. 그 결과 두 개의 서로 다른 밴드유형을 관찰하였으며, 21개 부위에서 염기서열 변이가 관찰되었다. 이러한 결과는 유전적 다양성 변이를 검출하는데 SSCP 분석이 유용한 도구라고 사료된다.

• Journal of Animal Science and Technology
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• 제47권2호
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• pp.159-166
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• 2005

Porcine myostatin(MS1N) gene plays a key role in the differentiation of myoblast and muscle development. Genetic polymorphism was screened by single stranded conformation polymorphism(SSCP) analysis and subsequent DNA sequencing detected a nucleotide substitution(C2150T) in exon 3 of MSIN gene. Phenotypic association of the polymorphism was tested in a Landrace population and positive effects of the allele T for lean growth traits were found in the population. Even though it is not significant, the pigs have IT and TC genotypes were heavier for the body weight at birth and at twenty weeks of age than those containing genotype. Cc. However, the allele T was significantly associated with higher eye muscle area(P < 0.05). As a result of this study, we suggested that the allele T in exon 3 of MSTN gene comes a significant effect for increasing the eye muscle area without decreasing backfat thickness. This polymorphism did not change the amino acid but Taq I -RFLP matched to SSCP band patterns in exon 3 of MSTN gene, which will be an useful molecular marker for breeding of Landrace pigs.

• 한국동물학회:학술대회논문집
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• 한국동물학회 2003년도 한국생물과학협회 학술발표대회
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• pp.210.2-210
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• 2003

No Abstract, See Full Text

• Asian-Australasian Journal of Animal Sciences
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• 제29권3호
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• pp.315-320
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• 2016

Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5'-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.