• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1863-1869
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• 2014

The amplification of telomerase component (TERC) gene could play an important role in generation and treatment of haematological malignancies. This present study was aimed to investigate copy number amplification status of TERC gene in chronic myeloid leukaemia (CML) patients who were being treated with imatinib mesylate (IM). Genomic DNA was extracted from peripheral blood of CML-IM Resistant (n=63), CML-IM Respond (n=63) and healthy individuals (n=30). TERC gene copy number predicted (CNP) and copy number calculated (CNC) were determined based on $Taqman^{(R)}$ Copy Number Assay. Fluorescence in situ hybridization (FISH) analysis was performed to confirm the normal signal pattern in C4 (calibrator) for TERC gene. Nine of CML patients showed TERC gene amplification (CNP=3), others had 2 CNP. A total of 17 CML patients expressed CNC>2.31 and the rest had 2.31>CNC>1.5. TERC gene CNP value in healthy individuals was 2 and their CNC value showed in range 1.59-2.31. The average CNC TERC gene copy number was 2.07, 1.99 and 1.94 in CML-IM Resistant patients, CML-IM Respond and healthy groups, respectively. No significant difference of TERC gene amplification observed between CML-IM Resistant and CML-IM Respond patients. Low levels of TERC gene amplification might not have a huge impact in haematological disorders especially in terms of resistance towards IM treatment.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.17
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• pp.7997-8002
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• 2015

Gene amplification is an important mechanism in the development and progression of cancer. Currently, gene amplification status is generally determined by in situ hybridization (ISH). Multiplex ligation-dependent probe amplification (MLPA) is a PCR-based method that allows copy number detection of up to 50 nucleic acid sequences in one reaction. The aim of the present study was to compare results for HER2, CCND1, MYC and ESR1 gene amplification detected by MLPA with fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) as clinically approved methods. Tissue samples of 170 invasive breast cancers were collected. All were ER positive. Tissue samples had previously been tested for HER2 using immunohistochemistry. Amplification of the selected genes were assessed using MLPA, FISH and CISH and results were compared. HER2 MLPA and ISH results were also compared with HER2 immunohistochemistry (IHC) which detects protein overexpression. Amplification of HER2, CCND1, MYC and ESR1 by MLPA were found in 9%, 19%, 20% and 2% of samples, respectively. Amplification of HER2, CCND1, MYC and ESR1 by FISH was noted in 7%, 16%, 16% and 1% of samples, respectively. A high level of concordance was found between MLPA/FISH (HER2: 88%, CCND1: 88%, MYC: 86%, ESR1: 92%) and MLPA/CISH (HER2: 84%). Of all IHC 3+ cases, 91% were amplified by MLPA. In IHC 2+ group, 31% were MLPA amplified. In IHC 1+ group, 2% were MLPA amplified. None of the IHC 0 cases were amplified by MLPA. Our results indicate that there is a good correlation between MLPA, IHC and ISH results. Therefore, MLPA can serve as an alternative to ISH for detection of gene amplification.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.2
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• pp.693-697
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• 2015

Our aims were to evaluate the clinical performance of human telomerase RNA gene component (hTERC gene) amplification assay with high-risk human papillomavirus (HR-HPV) DNA test of Hybrid Capture 2 DNA test (HC2), for the detection of high grade cervical precancerous lesions and cancer (CIN 2+). In addition, the association shown between hTERC gene amplification and HPV DNA test positive in women with and without cervical neoplasia was assessed. There were 92 women who underwent cytology, HR-HPV DNA test, hTERC gene amplification test, colposcopy and biopsy. We compared the clinical performance of hTERC gene test along with HR-HPV DNA test of women with colposcopy and routine screening. The samples were histology-confirmed high-grade cervical intraepithelial neoplasia (CIN 2) or worse (CIN2+) as the positive criterion. The test of hTERC gene showed the hTERC gene amplification positivity increased with the severity of histological abnormality and cytological abnormality. The test of hTERC gene showed higher specificity than HR-HPV DNA test for high-grade lesions (84.4% versus 50%) and also higher positive predictive value (90.4% versus 76.5%). Our results predicted that hTERC gene amplification demonstrated more specific performance for predicting the risk of progression and offer a strong potential as a tool for triage in cervical cancer screening, with the limited sensitive as HR-HPV DNA test.

• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.544-549
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• 1995

The alkaline elastase is an extracellular serine protease of the alkalophilic Bacillus strain Ya-B. To increase the gene copy number and the production level of the alkaline elastase Ya-B, we designed, on the B. subtilis chromosome, a gene amplification of the 10.6 kb repeating unit containing amyE, aleE (alkaline elastase Ya-B gene) and tmrB. The aleE was inserted between amyE and tmrB, and B. subtilis APT119 strain was transformed with this amyE-aleE-tmrB-junction region fragment. As a result, we succeeded in obtaining tunicamycin-resistant (Tm$^{r}$) transformants (Tf-1, Tf-2) in which the designed gene amplification of 10.6 kb occurred in chromosome. The transformants showed high productivity of $\alpha $-amylase and alkaline elastase Ya-B. The copy number of the repeating unit (amyE-aleE-tmrB) was estimated to be 25, but plasmid vector (pUC19) was not integrated. The amplified aleE of chromosome was more stable than that of plasmid in absence of antibiotics.

• Genomics & Informatics
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• v.19 no.3
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• pp.30.1-30.8
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• 2021

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

• Fisheries and Aquatic Sciences
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• v.17 no.3
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• pp.339-344
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• 2014

PCR amplification with universal primer is a useful tool for speciation of symbionts in marine eukaryote coupled with robust separation method such as denaturing high performance chromatography (DHPLC). To overcome the biased amplification, clamping PCR is recommended to suppress the amplification of host gene. In this study, we evaluated the efficiency of rare gene detection for two kinds of clamping probes which were successfully utilized for eukaryotic symbiont analysis: C3 linked nucleotide (C3) and peptide nucleic acid (PNA). PNA was 3-4 orders of magnitude higher than that of C3 tested in clamping efficiency and rare gene detection. This represented that PNA could be a more competent clamping probe for the enhancement of PCR amplification for rare symbiont genes.

• Journal of Microbiology and Biotechnology
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• v.30 no.5
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• pp.662-667
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• 2020

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.26 no.4
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• pp.355-362
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• 2000

Neoplastic growth is characterized by alterations of oncogenes and antioncogenes. The interaction between activated oncogenes and functional deletion of antioncogene appears to be the driving force directing normal cells to uncontrolled growth resulting in tumor. In addition to those genes mentioned, other genes controlling the entry of cells into the cell cycle have recently been implicated in cancer development. The overexpression of the cyclin D1 gene, which has been mapped to 11q13, either by gene rearrangement or amplification has been noted in various malignant tumors. The product of the cyclin D1 gene forms a complex with cyclin-dependent protein kinases(CDK4) that governs a key transition in the cell cycle. The relationships between the overexpression of cyclin D1 assessed by immunihistochemistry and the amplification of the cyclin D1 gene by differential polymerase chain reaction(DPCR) using primers for dopamin D2 receptor gene in 13 cases of squamous cell carcinomas of the oral cavity have been studied. The semiquantitative assay of cyclin D1 amplification has been made by cyclin D1/dopamin D2 receptor(CD/DR) ratio. The results were as follows; 1. In the normal tissue and the tumor, the CD/DR ratios were 0.82 and 1.36 respectively. This implicates 1.65-fold amplification of cyclin D1 gene in tumor compared to that in normal tissue. 2. The tumor tissue which showed overexpression of cyclin D1 by immunohistochemistry revealed 2-fold amplification of cyclin D1 compared to the normal tissue. 3. The tumor tissue which showed mild expression of cyclin D1 by immunihistochemistry revealed 1.7-fold amplification of cyclin D compared to the normal tissue. 4. The cyclin D1 was overexpressed in the tumor tissue at the rate of 38%. Above results suggest that cyclin D1 has close correlation with the development of carcinoma in the oral cavity. But further studies were needed to elucidate the carcinogeneic mechanisms by comparative studies among cyclin D1, pRb and p53.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.117-127
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• 2001

The high-level expression of a human single chain urokinase-type plasminogen activator (scu-PA) was achieved by employing a methotrexate (MTX)-dependent gene amplification system in Chinese hamster ovary (CHO) cells. By cotransfecting and coamplifying a scu-PA expression plasmid and dihydrofolate reductase (DHFR) minigene, several scu-PA expressing CHO cell lines were selected and gene-amplified. These recombinant cell lines, NGpUKs, secreted a completely processed scu-PA of 54 kD and up to 60mg/L was accumulated in the culture medium when they were adapted to an optimal MTX concentration. Over 95% of the scu-PA expressed was secreted in the culture medium and identified having the proper function of a plasminogen activator when activated by plasmin. Based on a genomic Southern analysis, a representative subclone, MGpUK-5, exhibited MTX-dependent scu-PA gene amplification, plus the initial single-copy gene of scu-PA eventually turned into about 150 copies of the amplified gene of scu-PA after gradual adaptation to 2.0$\mu$M of MTX. Meanwhile, the transcripts kof the scu-PA gene increased, although -early saturation of transcription was identified at 0.1$\mu$M of MTX. The scu-PA production by the MGpUK-5 subclone also increased relative to the gene amplification and increased transcripts, however, the relationship was not linearly proportional. Accordingly, since the MGpUK cell lines expressed elevated levels of enzymatically active scu-PA, these cell lines could be applied to the largescale production of scu-PA.

• Journal of Oral Medicine and Pain
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• v.19 no.2
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• pp.205-219
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• 1994

Cigarette butts from 5 smokers were gathered and then, placed in room temperature for 1, 3, 5, 7, 15 days. The possible use of the cigarette butts for individual identification was evaluated in sex determination, amplification of D1S80 locus, polymorphisms of HLA-DQA1 gene from the extracted DNA. 1. DNA extraction was possible in cigarette butts weree left in room temperature for 15days, so it can be applicatable to individual identification by polymerase chain reaction(PCR). 2. Amplification of X-Y homologous amelogenin gene by PCR made it possible to identify the sex in saliva stains (cigarette butts). 3. Amplification of D1S80 locus can be acquired from adding the boving serum albumin and hot start PCR procedures from forensic samples such as saliva stains (cigarette butts), so the AMP-FLPs examining is possible. 4. Genotype could be determined simply and rapidly using Amplitype$TM$ HLA-DQ$\alpha$ forensic kit in examining the HLA-DQA1 gene. From the investigation, DNA extraction, sex determination, amplification of D1S80 locus, polymorphisms of HLA-DQA1 gene was successfully done even though the cigarette butts were left for 15 days at room temperature. Therefore cigarette butts are highly reliable and applicatable as molecular biologic samples for individual identification.