• Journal of Animal Science and Technology
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• v.49 no.5
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• pp.549-558
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• 2007

C4B and BAT2, assigned to the SLA class III region, were recently reported on relation with human diseases. The primers for RT-PCR and RACE-PCR for CDS analysis of these genes of pig were designed by aligning the CDSs of humans and mice from GenBank. After we amplified and sequenced with these primers and cDNAs, the full-length CDSs of pig were determined. The CDS lengths of C4B and BAT2 were shown as 5226 bp and 6501 bp. In addition, the identities of nucleotide sequences with human and mouse were 76% to 87%, and the identities of amino acids were 72% to 90%. After we carried out the alignment with determined CDSs in this study and pig genomic sequences from GenBank, the primers for cSNP detection in genome were designed in intron regions that flanked one or more exons. Then, we amplified and directly sequenced with genomic DNAs of six pig breeds. Four cSNPs from C4B and three 3 cSNPs from BAT2 were identified. In addition, amino acid substitution occurred in six cSNP positions except for C4248T of C4B. By the Multiplex-ARMS method, we genotyped seven cSNPs with DNA samples used for direct sequencing. We verified that this result was the same as that analyzed using direct sequencing. To demonstrate recrudescence, we performed both direct sequencing and Multiplex-ARMS on two randomly selected DNA samples. The genotype of each sample showed the same result from both methods. Therefore, seven cSNPs were identified from C4B and BAT2 and could be used as the basic data for haplotype analysis of SLA class III region. Moreover, the Multiplex-ARMS method should be powerful for genotyping of genes assigned to the whole SLA region for the xenograft study.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.7
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• pp.2707-2712
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• 2015

Background: The human homologue of the mouse double minute 2 (MDM2) gene is a negative regulator of Tp53. MDM2-309T>G a functional promoter polymorphism was found to be associated with overexpression thereby attenuation of Tp53 stress response and increased cancer susceptibility. We have planned to evaluate the possible role of MDM2-309T>G polymorphism with risk and response to chemotherapy in AML. Materials and Methods: A total of 223 de novo AML cases and 304 age and sex matched healthy controls were genotyped for the MDM2-309T>G polymorphism through the tetra-primer amplification refractory mutation system (ARMS)-PCR method. In order to assess the functional relationship of -309T>G SNP with MDM2 expression level, we quantified MDM2 mRNA in 30 primary AML blood samples through quantitative RT-PCR. Both the (-309T>G) genotypes and the MDM2 expression were correlated with disease free survival (DFS) rates among patients who have achieved complete remission (CR) after first induction chemotherapy. Results: MDM2-309T>G polymorphism was significantly associated with AML development (p<0.0001). The presence of either GG genotype or G allele at MDM2-309 confered 1.79 (95% CI: 1.12-2.86; p<0.001) and 1.46 fold (95%CI: 1.14-1.86; p= 0.003) increased AML risk. Survival analysis revealed that CR+ve cases with GG genotype had significantly increased DFS rates (16months, p=0.05) compared to CR+ve TT (11 months) and TG (9 months) genotype groups. Further, MDM2 expression was also found to be significantly elevated in GG genotype patients (p=0.0039) and among CR+ve cases (p=0.0036). Conclusions: The MDM2-309T>G polymorphism might be involved in AML development and also serve as a good prognostic indicator.

• International Journal of Industrial Entomology and Biomaterials
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• v.45 no.1
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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.

• Journal of Animal Science and Technology
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• v.50 no.6
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• pp.753-762
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• 2008

The primers for RT-PCR and RACE-PCR were designed by aligning the pig genomic sequence and the human complement factor B(CFB) coding sequence(CDS) from the GenBank. Each PCR product was amplified in pig cDNA and sequencing was carried out. The CDS length of pig CFB gene was determined to be 2298 bp. In addition, the pig CDS was more longer than human and mouse orthologs because of insertion and deletion. The identities of porcine nucleotide sequences with those of human and mice were 84% and 80%, and the identities of amino acids were 79% to 77%, respectively. Three complement control protein(CCP) domains, one Von Willebrand factor A(VWFA) domain and a serine protease domain, that are revealed typically in mammals, were found in the pig CFB gene. Based on the CDSs determined, the primers were designed in intron regions for amplification of entire length of exons. In amplification and direct sequencing with genomic DNAs of six pig breeds, three cSNPs(coding single nucleotide polymorphisms) were identified and verified as missense mutations. Using the Multiplex-ARMS method, we genotyped and verified the mutations identified from direct sequencing. To demonstrate recrudescence, we performed both direct sequencing and Multiplex-ARMS with two randomly selected DNA samples. The genotype of each sample exhibited the same results using both methods. Therefore, three cSNPs were identified from pig CFB gene and that can be used for haplotype analysis of the swine leukocyte antigen(SLA) class III region. Moreover, the results indicate that the Multiplex-ARMS method should be powerful for genotyping of genes in the SLA region.

• BMB Reports
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• v.38 no.5
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• pp.555-562
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• 2005

Single nucleotide polymorphisms (SNPs) are becoming the most common type of markers used in genetic analysis. In the present report a SNP has been chosen to test the applicability of Real Time PCR to discriminate and quantify SNPs alleles on DNA pools. Amplification Refractory Mutation System (ARMS) and Mismatch Amplification Mutation Assay (MAMA) has been applied. Each assay has been pre-validated testing specificity and performances (linearity, PCR efficiency, interference limit, limit of detection, limit of quantification, precision and accuracy). Both the approaches achieve a precise and accurate estimation of the allele frequencies on pooled DNA samples in the range from 5% to 95% and don't require standard curves or calibrators. The lowest measurement that could be significantly distinguished from the background noise has been determined around the 1% for both the approaches, allowing to extend the range of quantifications from 1% to 99%. Furthermore applicability of Real Time PCR assays for general diagnostic purposes is discussed.

• Journal of Life Science
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• v.24 no.2
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• pp.181-185
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• 2014

We identified SNPs (single nucleotide polymorphisms) for endothelial nitric oxide synthase (eNOS) genes in the Korean genome. eNOS is present in the vascular endothelium, platelets, and several other cell types that continuously produce modest amounts of NO. Endothelium-derived NO plays a key role in the regulation of vascular tone, and the impaired effects of NO on the cardiovascular system appear to be responsible for coronary atherosclerosis and thrombosis. In recent studies, a missense variant within exon 7 of the eNOS gene in patients with coronary spastic angina-GAG to GAT substitution, which results in the replacement of glutamic acid by aspartic acid (Glu298Asp [G894T])-has been identified and is known to be significantly associated with coronary spasm. We prepared PCR primers based on sequences in Genbank. Primers were prepared for normal and SNPs separately, as reported for other Asian countries, such as G894T. Their sequences were different only at the 3' ends so that primer extension could only by possible when base pairs between templates and primers matched. We also employed ARMS (Amplification Refractory Mutation System) technology to improve the specificity of the PCR reaction. In conclusion, we were able to demonstrate the eNOS G894A polymorphism in Korean gemone. This study should facilitate research on the cause of myocardial infarction and development on further therapy at the genetic level.

• Journal of Sericultural and Entomological Science
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• v.42 no.1
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• pp.14-23
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• 2000

The mitochondrial genome of domesticated silkworm (Bombyx mori) was mapped with five restriction endonucleases (BamHI, EcoRI, HindIII, PstI and XbaI), the entire genome was cloned with HindIII and EcoRI. From the end sequencing results of 5$^1$and 3$^1$region for full genome set of eleven mitochondrial clones, the seven mitochondrial genes (NADH dehydrogenase 6, ATPase 6, ATPase 8, tRN $A^{Lys}$, tRN $A^{Asp}$, tRN $A^{Thr}$ and tRN $A^{Phe}$ of mori were identified on the basis of their nucleotide sequence homology. The nucleotide composition of NADH dehydrogenase 6 was heavily biased towards adenine and thymine, which accounted for 87.76%. On basis of the sequence similarity with published tRNA genes from six insect species, the tRN $A^{Lys}$, tRN $A^{Asp}$ and tRN $A^{Thr}$ were showed stable canonical clover-leaf tRNA structures with acceptible anticodons. However, both the DHU and T$\psi$C arms of tRN $A^{Phe}$ could not form any stable stem-loop structure. The two overlapping gene pairs (tRN $A^{Lys}$ -tRN $A^{ASP}$ and ATPase8-ATPase6) were found from our sequencing results. The genes are encoded on the same strad. ATPase8 and ATPase6 overlaps (ATGATAA) which are a single example of overlapping events between abutted protein-coding genes are common, and there is evidence that the two proteins are transcribed from a single bicistronic message by initiation at 5$^1$terminal start site for ATPase8 and at an internal start site for ATPase6. Ultimately, this result will provide assistance in designing oligo-nucleotides for PCR amplification, and sequencing the specific mitochondrial genes for phylogenetics of geographic races, genetically improved silkworm strains and wild silkworm (mandarina) which is estimated as ancestal of domesticated silkworm.sticated silkworm.

• Clinical and Experimental Pediatrics
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• v.46 no.5
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• pp.467-473
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• 2003

Purpose : The Epstein-Barr virus(EBV), gamma herpesvirus, is an important pathogen that is widespread around the world. The EBV causes various diseases depending on the geographic location, and on the immunity or the premorbid condition of the person exposed to EBV. To evaluate EBV typing may be the most important step to figure out the pathogenesis of EBV associated diseases, and we need to re-evaluate the pathologic role of human leukocyte antigen(HLA) in developing Epstein- Barr virus associated acute infectious mononucleosis by using newly developed methods. Methods : This study included 24 children(age range : 6 to 13 years), serologically confirmed with acute infectious mononucleosis. The control group for the HLA type consisted of 200 age-matched healthy children. To classify HLA I, modified ARMs-PCR was used, while modified PCR-SSOP was utilized in typing of HLA II. Also, we performed EBV typing in study patients by using a one-step PCR. Results : The results of HLA types : In HLA class I, HLA-A24 was positive in 69 of 200 healthy children and positive in 14 of 24 patients in the study group(relative risk : 3.5724, chi-square; 5.26, P<0.05). In HLA class II, HLA-DRB1*07 was detected in 18 of 200 healthy children, and eight of 24 patients in the study group(relative risk; 506173, chi-square; 9.73, P<0.01). The results of EBV types : In the research group, 20(83.8%) of 24 patients were shedding type A virus, while 4(16.7%) were type B. Conclusion : We conclude that development of infectious mononucleosis may be associated with HLA types, and these results suggest that acute infectious mononucleosis could have hereditary traits. And we confirm that type A EBV is highly prevalent in patients with acute infectious mononucleosis in Korea. Also, our results suggest that further large scale studies, including adult groups, regarding the association between pathogenesis of EBV with HLA-DP or HLA-DQ will be warranted.

• Genomics & Informatics
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• v.18 no.1
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• pp.9.1-9.5
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• 2020

Ulcerative colitis is a form of inflammatory bowel disease characterized by chronic inflammation of the colon and rectum. The abnormal lesions in the digestive system caused by ulcerative colitis and intermittent colitis are of major clinical importance. MDM2 is a phospho-protein that functions as a ubiquitin ligase for p53. Recently, a T>G substitution in the promoter of the MDM2 gene (rs309) has been identified. In this case-control study, 174 ulcerative colitis biopsy samples and 82 control samples were collected from colonoscopy centers, hospitals, and clinics in Mazandaran and Gilan Provinces in Iran from October 2014 to May 2015. This MDM2 polymorphism was investigated in DNA samples (extracted from biopsy samples) by amplification-refractory mutation system polymerase chain reaction. The mean age of patients with ulcerative colitis was 46.5 years (range, 28 to 69 years) and that of control individuals was 45.3 years (range, 26 to 71 years). Seventy-eight patients (44.82%) were men and 96 (55.18%) were women. The distribution of the TT, TG, and GG genotypes was 17.93%, 27.59%, and 34.48%, respectively, in the ulcerative colitis patients and 31.70%, 24.40%, and 43.90%, respectively, in the control individuals (odds ratio of GG for ulcerative colitis, 7.142; 95% confidence interval, 2.400 to 9.542; p = 0.001). It was found that a single-nucleotide polymorphism at rs309 in the MDM2 gene was associated with ulcerative colitis. A direct relationship was found between age and ulcerative colitis, while no relationship was found with sex. This finding is of note because the occurrence of intestinal inflammation and subsequent ulcers can precede the development of cancer.