단일모발로부터 DNA의 유전자형 검색

明賢君,金暻勳,黃迪駿 大韓法醫學會 1993 대한법의학회지 Vol.17 No.2

Hairs are one of the biological evidences which are found frequently at crime scenes, so their identification can be of considerable forensic importance. DNA were extracted from shaft as well as root of a single hair for DNA typing. The root end of a plucked single hair contains 120-200 ng DNA, whereas hair shaft contains too little DNA to determine its quantity. Using the polymerase chain reaction(PCR), genotypes of HLA DQA1 locus and AMP-FLPs(amplified fragment length polymorphisms) of D1S80 and COL2A1 locus were determined from hair shaft and hair root.

韓國人에서 VNTR D1S80 遺傳座位의 遺傳的 多樣性 및 集團의 均質性 檢定

明賢君,黃迪駿,洪鎔杓,金致弘 大韓法醫學會 1994 대한법의학회지 Vol.18 No.1

Human genomic DNA prepared from whole blood sample was analyzed to estimate allelic frequencies and genetic diversity at a hypervariable variable number of tandem repeats(VNTR) D1S80 locus in 127 Korean. Target DNA fragments were amplified by polymerase chain reaction(PCR) and separated by polyacrylamide gel electrophoresis. Then, the following results were obtained. 1. A total of 23 alleles were identified at this locus, which is the biggest number of alleles observed at this locus so far(cf. 16 from U. S. Caucasians and 15 from Finnish). 2. Sixty-two genotypes, included 3 homozygotes and 59 heterozygotes, out of a total of 276 probable genotypes were also observed, which might be resulted from the small sample size compared to the number of alleles observed in this study. 3. General pattern of allelic distributions was concordant with those observed in previous studies from Finnish and U. S. Causccasians where two alleles, M18 and M23, were observed most frequently and the rest of them were observed rare(<10%) with the exception of M30 allele(14.6%) in Korean. The number of M30 allele is suspected to be increased in Korean at or after the divergence of Korean from the hypothetical ancestor probably caused by random genetic drift such as founder effect of bottleneck effect. 4. Higher level of heterozygosity was obtained in Korean(H=0.866 and H=0.881) than in Finnish(H=0.77 and H=0.79) and U. S. Cauccasians(H=0.808 and H=0.797). 5. Population homogeneity was tested to corroborate the obtained population genetic parameters calculated based on the assumption of Hardy-Weinberg equilibrium, which could be done by Chi-square test between the observed number of alleles and the expected number of alleles, because of the small sample size analyzed in this study which resulted in high proportion of undetected genotypes (78.5%) and several genotypes significantly deviated from hardy-Weinberg expectation in a sample population. The result suggests that samples were drawn from geneticically homogeneous population. 6. This study suggests that Korean is more diversified than Finnish and U. S. Cauccasians on the basis of the number of alleles and the level of heterozygosity, although Korean is known to be entirely composed of single race. Though we need more informations from other Asian countries, this result suggests frequent gene flow from neighbor countries, which is also expected based on the old Korean history of frequent invasions from Japan and China. 7. Based on the Hardy-Weinberg expectation, 216 out of 253 genotypes at D1S80 locus can be shared by less than 5 individuals among 1,000 unrelated individuals by chance. Our results suggest that the analysis of VNTR D1S80 locus can provide powerful, but imperfect by itself, DNA markers for forensic study, which can be corroborated by analyzing additional VNTR loci.

Urea 젤 전기영동에 의한 CheY 아세틸화의 분석

배성호,명현군,박찬규,Bae, Seong-Ho,Myeong, Hyeon-Kun,Park, Chan-Kyu 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

Escherichia coli의 주화성 단백질인 CheY를 Affi-gel blue affinity chromatography와 Sephacryl S-200 gel filtration chromatography를 통하여 순수 분리하였다. 순수 분리된 CheY단백질은 효모로부터 분리한 acetyl-CoA synthetase(ACS)에 의해 in vitro에서 아세틸화 반응이 일어나는 것을 방사선 동위원소 표식법으로 확인하였다. 이 때 acetyl-CoA를 넣어주면 아세틸화가 줄어드는 것으로 보아 아세틸 공여체로 acetyl-CoA가 아닌 다른 물질이 이용되는 것으로 추측된다. Histone 단백질의 modification을 분석하기 위한 acid-urea gel을 변형하여 CheY의 분석에 적합한 urea gel을 고안하였다. Urea gel은 native gel에 비해 분해능이 더 좋고 CheY가 다른 단백질들과 뚜렷하게 분리되는 장점이 있었다. Urea gel을 사용하여 CheY의 아세틸화뿐만 아니라 CheA에 의한 인산화도 관찰하였으며, gel상에서 이들을 수식되지 않은 형태의 CheY로부터 분리할 수 있었다. CheY 단백질 종을 분리하여 분석한 결과 적어도 두 군데 이상의 아미노산 잔기에 수식이 일어날 가능성이 제시되었다. The CheY, a signaling protein involved in bacterial chemotaxis, was purified by Affi-gel blue affinity chromatography and Sephacryl S-200 gel filtration chromatography. Purified CheY was acetylated in vitro by acetyl-CoA synthetase (ACS; isolated from yeast) using $[2-^{14}C]-acetate$. Coenzyme A reduced the level of acetylated CheY, suggesting that other metabolic intermediates of acetate but not acetyl-CoA serves as an acetyl donor. To investigate the modification of CheY protein by acetylation, we developed a urea gel electrophoresis system similar to acid-urea gel electrophoresis that has been used for the analysis of histone modification. The resolution of urea gel was found better than that of native gel, in which CheY protein was clearly separated from other cellular proteins. In addition to CheY acetylation, CheY phosphorylation by CheA was detected on urea gel as multiply modified forms separated from the unmodified one. Analysis of the modified CheY bands revealed that CheY has more than one site for acetylation and for phosphorylation.

韓國人에서 重合酵素反應으로 檢索되는 COL2A1 遺傳座位의 對立遺傳子 頻度

黃迪駿,明賢君,李羲碩,郭明宰 大韓法醫學會 1994 대한법의학회지 Vol.18 No.2

With the human genomic DNAs obtained from 128 unrelated Korean, the VNTR region 3' to the collagen type Ⅱ gene(COL2A1) was amplified in vitro by the polymerase chain reaction(PCR), and separated by 3% agarose(2% Nusieve and 1% Seakem) and 4% polyacrylamide gel' and analyzed to get the population genetic data for COL2A1 VNTR allele such as allele and genotype frequencies, and hetrozygosity were calculated based on the amplified fragments length polymorphism(AMP-FLP). 1. In 128 unrdlated Korean, 4 allelic variants(K2, K3, K4, and K5) at COL2A1 VNTR locus are observed, but K1 allele which was also observed in other study was not. 2. The observed allele frequencies for K4, K5, k3 and k2 are 0.519, 0.383, 0.094 and 0.008, respectively. 3. Of 10 possible genotypes, only 4 heterozygous genotypes(K5/K4, K5/K3, k4/K3, K4/K2) and 2 homozygous genotypes(K5/K5, K4/K4) are observed. However, the observed numgers of homozygous and heterozygous genotypes(46 and 82, respectively) are not significatly different from their expectations, 53.3 and 74.7. 4. The observed genotype frequencies are ordered as K5/K4(0.445), K4/K4(0,242), K5/K5(0.117), K5/K3(0.086) and K4/K2(0.086), which are in accordance with the Hardy-weinberg prediction based on the observed allele frequencies. 5. The overall heterozygosity at this locus is 0.563, while its expectation based on the allele frequencies is 0.584

pV47-2 多座位 探針을 利用한 親生子鑑定

李卿,明賢君,鄭宰安,黃迪駿 大韓法醫學會 1993 대한법의학회지 Vol.17 No.1

DNA fingerprinting prepared using multi-locus DNA probe can provide valuable testing system in cases of disputed parentage. Because DNA-based testing takes the advantage of limitless numbers of VNTRs loci and the highly informative character of these markers. A simple approach based on Bayesian Theorem is described to the interpretation of the DNA fingerprints in disputed paternity cases that are detected by pV47-2 multilocus probe, including a Korean woman who has sought her biological father to a family consisting of a father and his two child. This paper then outline the analysis of each paternity case and give a numerical example as following results ; 1. From band-sharing coefficient(X=0.18) of DNA fingerprints detected by pV47-2 multilocus probe, the probability(r) that a parent will pass on a given DNA fingerprint to an offspring is 0.525. 2. As the biological mother(M), child(C), and alleged father(AF), or random man(RM) exhibit a given DNA fingerprint, paternity index(PI) is 1.298. 3. As alleged father or random man and Child share a given DNA fingerprint, but not M, PI is 5.526. 4. As the biological mother and child share a given DNA fingerprint, but not alleged father of random man, PI is 0.885. 5. As alleged father or random man only shares a given DNA fingerprint, PI is 0.525, whereas the biological mother only has one, PI is 1.1. 6. As the biological mother and alleged father or random man share a given DNA fingerprint, but not child, PI is 0.526. 7. When the biological mother, child and alleged father or random man do not share a given DNA fingerprint, PI is 1.1. 8. From the above paternity indices, likelihood ratio(LR) in case 1 is 99.994%, LR for child 1 and 2 in case 2 is 99.97% and 99.999%, respectively, but LR for unknown female in case 3 is 62,589% whereas LR for child 1 and 2 is 99.996% and 99.928%, respectively.

Urea 젤 전기영동에 의한 CheY 아세틸화의 분석

배성호,명현군,박찬규 ( Seong HO,Bae Hyeon Koon Chan Kyu Park ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

The CheY, a signaling protein involved in bacterial chemotaxis, was purified by Affi-gel blue affinity chromatography and Sephacryl S-200 gel filtration chromatography. Purified CheY was acetylated in vitro by acetyl-CoA synthetase (ACS; isolated from yeast) using [2-^(14)C]-acetate. Coenzyme A reduced the level of acetylated CheY, suggesting that other metabolic intermediates of acetate but not acetyl-CoA serves as an acetyl donor. To investigate the modification of CheY protein by acetylation, we developed a urea gel electrophoresis system similar to acid-urea gel electrophoresis that has been used for the analysis of histone modification. The resolution of urea gel was found better than that of native gel, in which CheY protein was clearly separated from other cellular proteins. In addition to CheY acetylation, CheY phosphorylation by CheA was detected on urea gel as multiply modified forms separated from the unmodified one. Analysis of the modified CheY bands revealed that CheY has more than one site for acetylation and for phosphorylation.

Whole genome amplification을 이용한 식중독 세균 신속 검출 기술 개발

성지영(Ji-Yeong Seong),고영준(Young-Jun Ko),명현군(Hyeon-Koon Myeong),오세욱(Se-Wook Oh) 한국식품과학회 2016 한국식품과학회지 Vol.48 No.2

PEG를 이용하여 WGA 수행 시 DNA 증폭 효율을 높이고 이를 식중독 세균의 DNA 증폭 및 검출에 적용하고자 하였다. 등온 증폭 반응인 WGA에 여러 종류의 PEG를 첨가하여 증폭한 결과, 1.5% 농도의 PEG 4,000을 첨가하는 것이 가장 효율이 높음을 알 수 있었다. 증폭 정도를 정량적으로 파악하기 위하여 3종의 식중독 세균 DNA를 이용하여 WGA를 수행하였으며 real-time PCR로 정량분석하였다. S. Typhimurium, L. monocytogenes, V. parahaemolyticus의 경우에 WGA를 하지 않은 DNA에 비하여 각각 7,777.01배, 9,981.22배, 1,239.03배 정도로 DNA의 양이 증폭되는 것을 확인하였다. 또한 PEG를 첨가함으로써 18배에서 40배의 핵산 증폭 효과가 더 있음을 알 수 있었다. 따라서 식품에 미량의 농도로 존재하는 식중독 세균은 PEG가 첨가된 WGA 반응을 통하여 검출 가능성을 높일 수 있음을 알 수 있었다. In this study, polyethylene glycol (PEG) was used to improve DNA amplification efficiency during whole genome amplification (WGA). Amplification efficiency was determined by adding PEG with different molecular weights to the WGA reaction. The greatest increase in amplification efficiency was obtained with PEG 4,000 used at 1.5% concentration. Foodborne pathogenic DNA was amplified by WGA and quantitatively analyzed by real-time polymerase chain reaction. DNA of Salmonella serotype Typhimurium, Listeria monocytogenes, and Vibrio parahaemolyticus was amplified 7,777.01, 9,981.22, and 1,239.03 fold, respectively, by WGA. On adding PEG in the WGA reaction (i.e., enhanced WGA [eWGA]), 18-40-fold more DNA amplification was achieved. Thus, these analyses showed that foodborne pathogens, which are usually present at very low concentration in foods, can be detected by real-time PCR and WGA.

수산물에서 식중독 세균을 신속하게 검출하는 최근 기술

김진희 ( Jin Hee Kim ),구은정 ( Eun Jeong Koo ),성지영 ( Ji Yeong Seong ),명현군 ( Hyeon Koon Myeong ),오세욱 ( Se Wook Oh ) 한국식품위생안전성학회 2016 Safe Food Vol.11 No.3