• Title/Summary/Keyword: Multiplex PCR

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The IL-1 Gene polymorphisms in Korean Children (한국인 아동에서의 IL-1 유전자 다변성 연구)

  • Yoon, Seong-Sik;Chung, Hyun-Ju;Kim, Ok-Su;Yang, Ku-Ho
    • Journal of Periodontal and Implant Science
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    • v.35 no.2
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    • pp.299-310
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    • 2005
  • The severe form of chronic periodontitis(CP) has been reported to be strongly associated with the presence of allele 2 of composite IL-1B(+3954) and IL-1A(+4845) genetic polymorphisms(genotype positive). However, other studies have reported conflicting findings. These might have resulted from differences in ethnic background and disease entities. The aim of this study was to determine the distribution of IL-1A(+4845), IL-1B(+3954), IL-1B(-511), and IL-1 RN(VNTR) genetic polymorphisms in children as a future Korean population. The study population consisted of 92 children from the Dept. of Pediatric Dentistry, Chonnam National University Hospital. Genomic DNA was obtained from buccal swab. The IL-1A(+4845), IL-1B(+3954), and IL-1B(-511) genes were genotyped by amplifying the polymorphic region using multiplex polymerase chain reaction(PCR), followed by restriction enzyme digestion and gel electrophoresis. IL-1 RN(VNTR) polymorphism were then evaluated by PCR amplification and fragment size analysis in agarose gel. The allele 2 frequency was 41.3%, 4.3%, 47.8%, and 9.9% for IL-1A(+4845), IL-1B(+3954), IL-1B(-511), and IL-1 RN respectively. The frequency of genotype with allele 2 carriage for IL-1A(+4845), IL-1B(+3954), IL-1B(-511), and IL-1 RN was 77.1%, 7.6%, 63.0%, and 15.2% respectively. The allele 2 frequency in IL-1B(+3954) was significantly higher in female than in male population(p<0.05). The negative association was shown between the presence of allele 2 in IL-1B(-511) and in IL-1B(+3954), and the carriage rate of IL-1B(+3954) allele 2 tended to lower in IL-1B(-511) allele 2(P=0.056). Only 7.3% of children carried the composite genotype of IL-1A(+4845) and IL-1B(+3954). These results suggest that the polymorphism of IL-1B(+3954) and the positive composite genotype was relatively rare in Korean population.

Genetic Polymorphism of Epoxide Hydrolase and GSTM1 in Chronic Obstructive Pulmonary Disease (만성폐쇄성폐질환 발생에 Epoxide hydrolase와 GSTM1유전자 다형성의 의의)

  • Park, Sang Sun;Kim, Eun Joung;Son, Chang Young;Wi, Jeong Ook;Park, Kyung Hwa;Cho, Gye Jung;Ju, Jin Young;Kim, Kyu Sik;Kim, Yu II;Lim, Sung Chul;Kim, Young Chul;Park, Kyung Ok;Na, Kook Joo
    • Tuberculosis and Respiratory Diseases
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    • v.55 no.1
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    • pp.88-97
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    • 2003
  • Background : Although smoking is a major cause of chronic obstructive pulmonary disease (COPD), only 10-20% of cigarette smokers develop symptomatic COPD, which suggests the presence of genetic susceptibility. This genetic susceptibility to COPD might depend on variations in the activities of the enzyme that detoxify hazardous chemical products, such as microsomal epoxide hydrolase (mEPHX) and glutathione-S transferase M1 subunit (GSTM1) genes. Methods : The genotypes of 58 patients with COPD, and 79 age matched control subjects, were determined by a polymerase chain reaction, followed by restriction fragment length polymorphism (PCR-RFLP) for the mEPHX, and multiplex PCR for the GSTM1. Results : GSTM1 was deleted in 53.3% of the subjects. There was no difference in GSTM1 deletion rates between the COPD patients (32/58, 55.2%) and the control subjects (41/79, 51.9%). The combination patterns of two polymorphisms of mEPHX showed slow enzyme activity in 29(21.2%), normal in 73(53.3%) and fast in 32(23.4%). The COPD group (7/57, 12.3%) showed a significantly lower incidence of slow enzyme activity compared to the control subjects (22/77, 28.6%, p<0.05). However, when the COPD and control groups were compared with smokers only, there were no significant differences in the genotypes of GSTM1 and mEPHX. Conclusion : The genotypes of GSTM1 and mEPHX were not significant risk factors of COPD in this cohort of study.

Polymorphisms of GSTM1 and CYP1A1, and Susceptibility to Primary Lung Cancer in Korean Males (한국인 남성에서 GSTM1과 CYP1A1 유전자 다형성과 원발성폐암의 유전적 감수성)

  • Bae, Nack-Cheon;Lee, Su-Yeon;Chae, Po-Hee;Kang, Kyung-Hee;Kim, Kyung-Rock;Cha, Seung-Ick;Chae, Sang-Chul;Kim, Chang-Ho;Jung, Tae-Hoon;Park, Jae-Yong
    • Tuberculosis and Respiratory Diseases
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    • v.50 no.5
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    • pp.568-578
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    • 2001
  • Background : Lung cancer is frequently cited as an example of a disease caused solely by exposure to environmental carcinogens. However, there is a growing realization that the genetic constitution is also important in determining individual's susceptibility to lung cancer. This genetic susceptibility may result from functional polymorphims of the genes involved in carcinogen metabolism. In this study, the association between GSTM1 and CYP1A1 polymorphisms and the lung cancer risk in Korean males was investigated. Materials and Method : The study population consisted of 153 male lung cancer patients and 143 healthy male controls. The GSTM1 and CYP1A1 genotypes were determined by multiplex PCR and PCR-RFLP analysis. Result : There were no significant differences in the frequency of the GSTM1 null genotype between the cases and the controls. When the cases were categorized by their histologic type, the frequency of the GSTM1 null genotype in the small cell carcinoma group was higher than those of the controls(67.2% vs 55.9%), but the difference was not statistically significant(OR=1.772 ; 95% CI=0.723-4.340). The distribution of the CYP1A1 MspI genotypes among the cases were similar to those among the controls. When the cases were grouped by their histologic type, the ml/m1, ml/m2, m2/m2 genotypes frequencies among the small cell carcinomas(23.0%, 38.5%, and 38.5%, respectively) were significantly different from those of the controls(36.4%, 46.2%, and 7.4%, respectively, p<0.05). When the m1/m1 genotype was used as a reference, the ml/m2 and m2/m2 genotypes were associated with an increased risk for small cell lung cancer(ml/m2 genotype : OR=1.337, 95% CI=0.453-3.947 ; m2/m2 genotype : OR=3.374, 95% CI=1.092-10.421). Conclusion : These results suggest that the GSTM1 and CYP1A1 genotypes may be a genetic determinant of the risk for lung cancer, particlulary small cell carcinoma. Further investigation is needed to confirm these results.

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Prevalence and Characterization of Virulence Genes in Escherichia coli Isolated from Diarrheic Piglets in Korea

  • Kim, Sung Jae;Jung, Woo Kyung;Hong, Joonbae;Yang, Soo-Jin;Park, Yong Ho;Park, Kun Taek
    • Journal of Food Hygiene and Safety
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    • v.35 no.3
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    • pp.271-278
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    • 2020
  • Enterotoxigenic Escherichia coli is one of the major causative infectious agents of diarrhea in newborn and post-weaning pigs and leads to a large economic loss worldwide. However, there is limited information on the distribution and characterization of virulence genes in E. coli isolated from diarrheic piglets, which also applies to the current status of pig farms in Korea. To investigate the prevalence and characterization of virulence genes in E. coli related to diarrhea in piglets, the rectal swab samples of diarrheic piglets (aged 2 d to 6 w) were collected from 163 farms between 2013 and 2016. Five to 10 individual swab samples from the same farm were pooled and cultured on MacConkey agar plates, and E. coli were identified using the API 32E system. Three sets of multiplex PCRs were used to detect 13 E. coli virulence genes. As a result, a total of 172 E. coli isolates encoding one or more of the virulence genes were identified. Among them, the prevalence of individual virulence gene was as follows, (1) fimbrial adhesins (43.0%): F4 (16.9%), F5 (4.1%), F6 (1.7%), F18 (21.5%), and F41 (3.5%); (2) toxins (90.1%): LT (19.2%), STa (20.9%), STb (25.6%), Stx2e (15.1%), EAST1 (48.3%); and (3) non-fimbrial adhesin (19.6%): EAE (14.0%), AIDA-1 (11.6%) and PAA (8.7%), respectively. Taken together, various pathotypes and virotypes of E. coli were identified in diarrheic piglets. These results suggest a broad array of virulence genes is associated with coliform diarrhea in piglets in Korea.

Analysis of Palivizumab Prophylaxis in Patients with Acute Lower Respiratory Tract Infection Caused by Respiratory Syncytial Virus (Respiratory syncytial virus로 인한 급성 하기도 감염 입원 환자에서 Palivizumab 예방요법 유무에 따른 비교 분석)

  • Min, Sung Ju;Song, Jung Sook;Choi, Jang Hwan;Seon, Han Su;Kang, Eun Kyeong;Kim, Do Hyun;Kim, Hee Sup
    • Pediatric Infection and Vaccine
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    • v.18 no.2
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    • pp.154-162
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    • 2011
  • Purpose : The aim of this study was to identify the clinical characteristics of lower respiratory tract infection due to respiratory syncytial virus (RSV) in young children and to provide information for an effective guideline for palivizumab administration in Korea. Methods : We reviewed medical charts of 167 patients under 3 years of age who were hospitalized in Dongguk University Ilsan Hospital for lower respiratory tract infection between January 2007 and February 2011. Diagnosis of the virus was made based on the multiplex real time polymerase chain reaction. Results : There were 113 patients who were infected by respiratory syncytial virus. 90 patients were term infants and 23 patients were preterm infants. No difference was shown between term and preterm infants except the days of admission which was 9.0${\pm}$6.0 days and 12.6${\pm}$21.0 days respectively. In the preterm group their mean age at the time of admission was 5.21${\pm}$4.9 months and the mean gestational age was 33.1${\pm}$4.3 weeks, and the mean birth weight was 2,152${\pm}$950 g. Only 4 patients were born under 28 weeks gestational age and were candidates for palivizumab administration. Conclusion : Most of the patients with severe RSV lower respiratory tract infection were term or near term infants who were not candidates for palivizumab prophylaxis. A nationwide study is needed to make a new risk stratified guideline for RSV prophylaxis for our country.

Development of SNP Markers for Domestic Pork Traceability (국내산 돼지고기의 원산지 검증을 위한 SNP Marker Set 개발)

  • Kim, Sang-Wook;Li, Xiaoping;Lee, Yun-Mi;Kim, Jong-Joo;Kim, Tae-Hun;Choi, Bong-Hwan;Kim, Kwan-Suk
    • Journal of Animal Science and Technology
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    • v.52 no.2
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    • pp.91-96
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    • 2010
  • The purpose of the study was to develop an optimum SNP marker set to be utilized for domestic pork traceability. The study tested 51 SNP markers analyzed for origin of farm to be determined from genotypes of offspring and parents in pigs. With the simulation data through random mating population (PI), half sib mating population ($PI_{half-sib}$) and full sib mating population ($PI_{sibs}$), probability of identical genotypes were analyzed as $5.63{\times}10^{-33}$, $4.35{\times}10^{-15}$ and $1.32{\times}10^{-15}$, respectively. The 51 SNP markers also had 100% accuracy for parental determination. These results suggest that if the pig breeding stock is genotyped with the 51 SNP markers, the genotype information of individual offspring can be checked for farm origins by tracing parental sow and sire. Therefore, these SNP markers will be useful to trace the pork from production to consumption in pigs.

Genomic Polymorphism Analysis using Microsatellite Markers in Gyeongju Donggyeong Dogs

  • Kim, Seung-Chang;Kim, Lee-Kyung;Choi, Seog-Kyu;Park, Chang-Min;Park, Sun-Ae;Cho, Yong-Min;Lim, Dajeong;Chai, Han-Ha;Lee, Seung-Hwan;Lee, Ji-Woong;Sun, Sang-Soo;Choi, Bong-Hwan
    • Reproductive and Developmental Biology
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    • v.36 no.4
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    • pp.243-248
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    • 2012
  • This study was conducted to find a useful marker for gene polymorphism analysis using Microsatellite marker (MS marker) in Gyeongju Donggyeong dog. Twenty three MS marker analyzed the genetic features of DNA using 100 Gyeongju Donggyeong dogs in Gyeongju area. It was performed multiplex PCR with 3 set primer divided 9, 10 and 4 by analysis of conditions among MS markers. The results were calculated heterozygosity, polymorphic information content (PIC), allele frequency and number of allele at each locus using Microsatellite Toolkit software and Cervus 3.0 program. Total 148 alleles were genotyped to determine and average 6.43 alleles was detected. FH3381 had the highest of 15 alleles and FH2834 had the lowest of 2 alleles. Expected heterozygosity had a wide range from 0.282 to 0.876 and had average value of 0.6496. Also, Observed heterozygosity had a more wide range from 0.200 to 0.950 and had average value of 0.6404. PIC had range from 0.262 to 0.859 and average PIC was calculated 0.606. Especially, FH2998 represented the highest rate of observed heterozygosity of 0.950 and FH3381 represented the highest rate of expected heterozygosity of 0.876 and PIC of 0.859. The use of these markers was considered to be useful to study genetic traits of Gyeongju Donggyeong dog.

Polymorphic Variation in Glutathione-S-transferase Genes and Risk of Chronic Myeloid Leukaemia in the Kashmiri Population

  • Bhat, Gulzar;Bhat, Ashaqullah;Wani, Aadil;Sadiq, Nida;Jeelani, Samoon;Kaur, Rajinder;Masood, Akbar;Ganai, Bashir
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.1
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    • pp.69-73
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    • 2012
  • Cancer is a complex disease and the genetic susceptibility to it could be an outcome of the inherited difference in the capacity of xenobiotic metabolizing enzymes. Glutathione-S-transferases (GSTs) are phase II metabolizing enzymes whose various genotypes have been associated with increased risk of different types of cancer. Null mutations caused by the deletion of the entire gene result in the absence of the enzymatic activity and increase in the risk of developing cancer including chronic myeloid leukaemia (CML). In the present case-control study we evaluated the effect of null mutations in GSTM1 and GSTT1 genes on the risk of developing CML. The study included 75 CML patients (43 males and 32 females; age (mean ${\pm}$ S.D) $42.3{\pm}13.4$ years) and unrelated non-malignant controls (76 male and 48 females; age (mean ${\pm}$ S.D) $41.5{\pm}12.9$). The distribution of GSTM1 and GSTT1 genotypes in CML patients and controls was assessed by multiplex-PCR method. Logistic regression was used to assess the relationship between GSTM1 and GSTT1 genotypes and risk of CML. Chi-square test was used to evaluate the trend in modulating the risk to CML by one or more potential high risk genotype. Although GSTM1 null genotype frequency was higher in CML patients (41%) than in the controls (35%), it did not reached a statistical significance (OD = 1.32, 95% CI: 0.73-2.40; P value = 0.4295). The frequency of GSTT1 null genotypes was higher in the CML patients (36%) than in the controls (21%) and the difference was found to be statistically significant (OD = 2.12, 95% CI: 1.12-4.02; P value = 0.0308). This suggests that the presence of GSTT1genotype may have protective role against the CML. We found a statistically significant (OD = 3.09, 95% CI: 1.122-8.528; P value = 0.0472) interaction between the GSTM1 and GSTT1 null genotypes and thus individuals carrying null genotypes of both GSTM1 and GSTT1 genes are at elevated risk of CML.

Characterization of a Novel cry1-Type Gene from Bacillus thuringiensis subsp. alesti Strain LY-99

  • Qi, Xu Feng;Li, Ming Shun;Choi, Jae-Young;Roh, Jong-Yul;Song, Ji Zhen;Wang, Yong;Jin, Byung-Rae;Je, Yeon-Ho;Li, Jian Hong
    • International Journal of Industrial Entomology and Biomaterials
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    • v.18 no.1
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    • pp.18-27
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    • 2009
  • B. thuringiensis strain LY-99 belonging to subsp. alesti (H3a3c), was isolated from Chinese tobacco warehouse and showed significantly high toxicity to Plutella xylostella. For the identification of the cry1-type genes from B. thuringiensis LY-99, an extended multiplex PCRrestriction fragment length polymorphism (PCRRFLP) method was established by using two pairs of universal primers based on the conserved regions of the cry1-type genes to amplify around 2.4 kb cry1-type gene fragments. Then the DNA fragment was cloned into pGEM-T Easy vector and digested with EcoRI and EcoRV enzymes. Through this method, a known cry1-type gene was successfully identified from the reference strain, B. thuringiensis subsp. alesti. In addition, the RFLP patterns revealed that B. thuringiensis LY-99 included a novel cry1A-type gene in addition to cry1Aa, cry1Ac, cry1Be and cry1Ea genes. The novel cry1A-type gene was designated cry1Ah2 (Genbank accession No DQ269474). An inverse PCR method was used to amplify the flank regions of cry1Ah2 gene. Finally, 3143 bp HindIII fragment from B. thuringiensis LY-99 plasmid DNA including 5' region and partial ORF was amplified, and sequence analysis revealed that cry1Ah2 gene from LY-99 showed 89.31% of maximum sequence similarity with cry1Ac1 crystal protein gene. In addition, the deduced amino acid sequence of Cry1Ah2 protein shared 87.80% of maximum identity with that of Cry1Ac2. This protein therefore belongs to a new class of B. thuringiensis crystal proteins.

Construction of a DNA Profile Database for Commercial Cucumber (Cucumis sativus L.) Cultivars Using Microsatellite Marker (Microsatellite 마커를 이용한 오이 유통품종 DNA Profile Data Base 구축)

  • Kwon, Yong-Sham;Choi, Keun-Jin
    • Horticultural Science & Technology
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    • v.31 no.3
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    • pp.344-351
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    • 2013
  • Microsatellite is one of the most suitable marker for cultivar identification as it has great discrimination power for cultivars with narrow genetic variation. The polymorphism level between 358 microsatellite primer pairs and 11 commercial cucumber cultivars was investigated. Thirty-one primer pairs showed high polymorphism within cucumber cultivars with different fruit types. These markers were applied for the constructing DNA profile data base of 110 commercial cucumber cultivars through multiplex PCR and fluorescence based automatic detection system. A total of 139 polymorphic amplified fragments were obtained by using 31 microsatellite markers. The average of PIC value was 0.610 ranging from 0.253 to 0.873. One hundred and thirty nine microsatellite loci were used to calculate Jaccard's distance coefficients for UPGMA cluster analysis. A clustering group of varieties, based on the results of microsatellite analysis, were categorized into plant shape and fruit type. Almost the cultivars were discriminated by marker genotypes. This information may be useful to compare through genetic relationship analysis between existing variety and candidate varieties in distinctive tests and protection of plant breeders' intellectual property rights through variety identification.