• Asian Pacific Journal of Cancer Prevention
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• 제15권17호
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• pp.7377-7381
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• 2014

CYP2E1 PstI polymorphism G-1259C (rs3813867) genotype distributions vary significantly among different populations and are associated with both diseases, like cancer, and adverse drug effects. To date, there have been limited genotype distributions and allele frequencies of this polymorphism reported in the three major indigenous ethnic groups (KadazanDusun, Bajau, and Rungus) in Sabah, also known as North Borneo. The aim of this study was to investigate the genotype distributions and allele frequencies of the CYP2E1 PstI polymorphism G-1259C in these three major indigenous peoples in Sabah. A total of 640 healthy individuals from the three dominant indigenous groups were recruited for this study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) at G-1259C polymorphic site of CYP2E1 gene was performed using the Pst I restriction enzyme. Fragments were analyzed using agarose gel electrophoresis and confirmed by direct sequencing. Overall, the allele frequencies were 90.3% for c1 allele and 9.7% for c2 allele. The genotype frequencies for c1/c1, c1/c2 and c2/c2 were observed as 80.9%, 18.8%, and 0.3%, respectively. A highly statistical significant difference (p<0.001) was observed in the genotype distributions between indigenous groups in Sabah with all Asian and non-Asian populations. However, among these three indigenous groups, there was no statistical significant difference (p>0.001) in their genotype distributions. The three major indigenous ethnic groups in Sabah show unique genotype distributions when compared with other populations. This finding indicates the importance of establishing the genotype distributions of CYP2E1 PstI polymorphism in the indigenous populations.

• Animal cells and systems
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• 제13권2호
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• pp.179-186
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• 2009

Discriminating between eel species of the genus Anguilla using morphological characteristics can be problematic, particularly in the glass eel and elver stages. In this study, sequence-characterized amplified region (SCAR) and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) markers were developed for the identification of Anguilla japoniea, Anguilla btcoior bicaor. Anguilla rostrata, and Anguilla anguilla. Random amplified polymorphic DNA (RAPD) fragments from A. japoniea (362 bp), A. bicolor bicctor (375 bp), A. rostrata (375 bp), and A. anguilla (375 bp) were isolated, sequenced, and converted to SCAR markers. The principal difference between the SCARs of A. japoniea and the three other species is the absence of a 13 bp deletion in the A. japoniea SCAR. Specific PCR primers amplified a 290 bp fragment for A. japoniea and 303 bp fragments for A. bicolor bicoior. A. rostrata, and A. anguilla. Restriction enzyme digestion with Taql, Mael, and Tru9l yielded PCR-RFLP patterns with differences that, when analyzed together, are sufficient for distinguishing each of the four eel species. In addition, RAPD fragments for A. japoniea (577 bp), A. bicoior bicoor (540 bp), A. rostrata (540 bp), and A. anguilla (509 bp) were also isolated and sequenced. The A. japoniea, A. bicoior blcoior. A. rostrata, and A. anguilla PCR products contain ten, nine, nine, and eight tandem repeats, respectively, of a 37 bp sequence. These results suggest that SCAR and PCR-RFLP markers and repeat numbers for specific loci will be useful for the identification of these four Anguilla eel species.

• Asian-Australasian Journal of Animal Sciences
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• 제19권2호
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• pp.165-170
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• 2006

Using the somatic cell hybrid panel (SCHP) and radiation hybrid (IMpRH) panel, porcine SOCS2 gene was mapped at SSC5 (1/2) q21-q24 and closely linked with SW1383 marker (47 cR in distance), while SOCS3 gene was assigned to SSC12p11-(2/3p13) and closely linked with SW2490 (43 cR). The reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect the expression of these two genes in the different tissues and the results showed that both SOCS2 and SOCS3 genes were widely expressed in tissues investigated (heart, liver, spleen, lung, kidney skeletal muscle, fat and brain), although some tissues showed lower gene expression. Moreover, SOCS2 and SOCS3 genes had different expression levels at different stages, in different tissues and in different breeds. A G/A substitution, which can be recognized by restriction enzyme of Cfr421, was observed in 5' untranslated region (5'-UTR) of SOCS2 gene. The allele frequencies was investigated by PCR-restriction fragment length polymorphism (PCR-RFLP) method and it showed that the allele frequency among Dahuabai, Erhualian, Yushan, Qingping, Large white and Landrace tested were different. Association analysis in a cross experimental populations revealed no significant association between the SOCS2 gene polymorphism and the economic traits investigated. The full-length coding regions (CDs) of porcine SOCS3 gene was obtained by RT-PCR.

• 대한임상검사과학회지
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• 제45권2호
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• pp.66-72
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• 2013

Fine Needle Aspiration Biopsy Cytology (FNABC), which is known as the most accurate and cost-effective method for diagnosis of the thyroid nodule, may still result in indeterminate cases that are cellular paucity and show minor nuclear atypia. However, most cases are associated with suspicion of papillary thyroid carcinoma (PTC). A B-type Raf kinase (BRAF) mutation was found in about half of PTCs which is currently helping us to differentiate malignancies from benign lesions. Cases studied included 46 histological, confirmed PTC cases. FNABC 102 cell paucity and 74 atypia benign cases were previously diagnosed as suspicious of PTC using cytologic examination. These cases were analyzed for BRAF mutation by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with a new restriction enzyme. In this study, the sensitivity and specificity were calculated and, BRAF mutation was detected by means of a histological method in 23 of 46 cases of PTC and no mutation was found in 22 cases. However, one case was not detected. In using FNABC, BRAF mutation was detected in 6 of 102 cases in cell paucity and in 11 of 74 cases in the atypia. Two cases were not detected in the atypia. The sensitivity and specificity of PCR-RFLP in FNABC were 60% and 97.4% respectively. Assessment of Formalin Fixed Paraffin Embedding (FFPE) block demonstrated similarly a 51.1% positive and 48.9% negative in PTC. Evaluation of BRAF mutation revealed high specificity and low sensitivity in using FNABC method. This study suggests that BRAF mutation analysis should be useful for the clinical diagnosis of PTC in FNABC with cytological findings suspicious for PTC.

• Archives of Pharmacal Research
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• 제29권1호
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• pp.88-95
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• 2006

Analysis of molecular nature of mitochondrial DNA (mtDNA) could be powerful marker for anthropological studies of modern populations. While population genetic studies on mtDNA have been reported for several ethnic groups, no such study has been documented for the Korean population. We surveyed mtDNA polymorphisms in the HVS I of noncoding D-loop region and its upstream region from 430 unrelated healthy Korean population by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing analysis. PCR product with 2,790 bp spanning the specific mtDNA region (mt13715-16504) was subjected to RFLP analysis using 6 restriction enzyme (Hinf I, Hae III, Alu I, Dde I, Mbo I, Rsa I). On the PAUP analysis of PCR-RFLP results, 38 mtDNA haplotypes (Hap 1-38) were detected in the Korean populations, which were classified into 11 haplogroups (Grp 1-11) of related haplotypes encompassing all 38 haplotypes. In comparison of sequencing data with Anderson's reference sequence, the transition type was more prevalent than the transversion type. Insertions or deletions were not found. In addition, three of the polymorphic sites (A16240C, A16351G, G16384A) in HVS-I region are determined newly. The polymorphic sites were distributed randomly in the region, though the frequency at each site was variable. Thus, this research might be required for the genealogical study of Orientals.

• ALGAE
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• 제21권2호
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• pp.261-266
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• 2006

The innate soluble polysaccharides and phenolic compounds of marine macroalgae are serious contaminants which interfere with experimental procedures such as restriction enzyme digestion, polymerase chain reaction (PCR) and other enzymatic reactions using extracted DNA samples. The viscous polysaccharides are co-precipitated with DNA samples by isopropanol or ethanol precipitation in conventional experiment. To overcome the problem, a method for the isolation of high molecular weight DNA from Porphyra tenera is developed with the application of diatomaceous earth column. The isolated DNAs by this method were about 50-100 kb in size and could be digested well with restriction enzymes. The nuclease activity seemed to be minimal, and high reproducibility in the arbitrary primed PCR for RAPD analyses was a distinctive feature. These results suggest that this method is very efficient in isolating nucleic acid from macroalgae including Porphyra.

• 대한의생명과학회지
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• 제8권3호
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• pp.107-113
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• 2002

Enterohemorrhagic Escherichia coli (EHEC) strains of serotype O157:H7 have been shown to colonize the intestinal epithelial cell by the attaching and effacing (AE) mechanism. The AE lesion is mediated by an intimin, of which production and expression are controlled by a 3-Kb eaeA gene located EHEC chromosomal DNA. If the eaeA gene is mutated, EHEC O157:H7 strains lose capacity of adhesion to intestinal epithelial cells. In this study, a 891 bp of the 3'-end region of a gamma intimin was amplified by polymerase chain reaction (PCR). The PCR product was inserted into pSTBlue-1 cloning vector and transformed into DE3 (BL21) competent cell. After plasmid mini-preparation and restriction enzyme digestion of eaeA/891-pSTBlue-1 vector, target eaeA gene was re-inserted into pET-28a expression vector and was transformed. Then the expression of recombinant eaeA/891 (891 bp) gene was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG). The expression of the 40-KDa recombinant protein was identified in SDS-PAGE and confirmed by immunoblotting using the His.Tag$^{\circledR}$ and T$_{7}$.Tag$^{\circledR}$ monoclonal antibody. This recombinant protein expressed by eaeA gene could be applied in further studies on the mechanisms of E. coli O157:H7 infection and the development of recombinant vaccine.

• Asian-Australasian Journal of Animal Sciences
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• 제28권11호
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• pp.1545-1550
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• 2015

c-MYC (v-myelocytomatosis viral oncogene homologue) is a transcription factor that plays important role in many biological process including cell growth and differentiation, such as myogenesis and adipogenesis. In this study, we aimed to detect MYC gene polymorphisms, their genotype frequencies and to determine associations between these polymorphisms and meat quality traits in Berkshire pigs. We identified a single nucleotide polymorphism (SNP) in intron 2 of MYC gene by Sanger sequencing, i.e., g.3350G>C (rs321898326), that is only found in Berkshire pigs, but not in other breeds including Duroc, Landrace, and Yorkshire pigs that were used in this study. Genotypes of total 378 Berkshire pigs (138 sows and 240 boars) were determined using Hha I restriction enzyme digestion after polymerase chain reaction. Observed allele frequencies of GG, GC, and CC genotypes were 0.399, 0.508, and 0.093 respectively. Statistical analysis indicated that the g.3350G>C polymorphism was significantly associated with $pH_{45min}$ and cooking loss (p<0.05), suggesting that g.3350G>C SNP can be used for pre-selection of $pH_{45min}$ and cooking loss traits in Berkshire pigs.

• Asian Pacific Journal of Cancer Prevention
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• 제16권2호
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• pp.485-488
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• 2015

Background: Oxidative stress caused by the generation of reactive oxygen species plays an important role in human carcinogenesis. Manganese superoxide dismutase (MnSOD) Val-9Ala in the mitochondrial target sequence is the best known polymorphism of this enzyme. The purpose of the current research was to assess the association of MnSOD Val-9Ala genotypes with the risk of gastric cancer. Materials and Methods: This case-control study covered 54 gastric cancer patients compared to 100 cancer free subjects as controls. Extraction of DNA was performed on bioptic samples and genotypes were identified with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results: The frequencies of MnSOD Ala/Ala, Ala/Val and Val/Val genotypes in healthy individuals were 24.3, 66.7 and 9%, respectively. However, in gastric cancer patients, Ala/Ala, Ala/Val and Val/Val were observed in 24.0, 48.0 and 28.0% (p=0.01). In patients the frequency of MnSOD Val allele was higher (52%) compared to that in controls (42%). Conclusions: The results of this study show a positive association between MnSOD Val-9Ala gene polymorphism and risk of gastric cancer disease in Iranian population.

• The Plant Pathology Journal
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• 제19권3호
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• pp.159-161
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• 2003

The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.