No abstract available.
*Gene Amplification ; *Gene Deletion ; *Polymorphism, Single Nucleotide ; Stomach Neoplasms/*genetics

OBJECTIVE: L and E6/E7 gene amplification analyses were compared to identify human papillomavirus (HPV) infection and verify the HPV type, with the intent to minimize HPV typing errors. METHODS: L1 gene verified HPV typing was accomplished via polymerase chain reaction (PCR) and membrane assays. Verification of HPV typing via E6/E7 genes was accomplished through nested multiplexed PCR. The results from 104 samples were compared. RESULTS: The rates of accordance and difference were 35% and 65%, respectively. For 29% of the analyses, nested multiplexed PCR was more diversified than the membrane assay. CONCLUSION: HPV can be classified into low-risk HPV and high-risk HPV groups. In parallel amplifications of the L and E genes is more efficient for accurate diagnosis in light of the different symptoms and attendant precautions of the risk groups.
Gene Amplification ; Humans ; Light ; Membranes ; Polymerase Chain Reaction

OBJECTIVE: L and E6/E7 gene amplification analyses were compared to identify human papillomavirus (HPV) infection and verify the HPV type, with the intent to minimize HPV typing errors. METHODS: L1 gene verified HPV typing was accomplished via polymerase chain reaction (PCR) and membrane assays. Verification of HPV typing via E6/E7 genes was accomplished through nested multiplexed PCR. The results from 104 samples were compared. RESULTS: The rates of accordance and difference were 35% and 65%, respectively. For 29% of the analyses, nested multiplexed PCR was more diversified than the membrane assay. CONCLUSION: HPV can be classified into low-risk HPV and high-risk HPV groups. In parallel amplifications of the L and E genes is more efficient for accurate diagnosis in light of the different symptoms and attendant precautions of the risk groups.
Gene Amplification ; Humans ; Light ; Membranes ; Polymerase Chain Reaction

Bacillus licheniformis alpha-amylase (BLA) is one of the most important enzymes involved in starch hydrolysis and many biotechnological processes. To improve the BLA productivity, an integrative plasmid pBL-amyL carrying amyL gene encoding a thermophilic alpha-amylase of B. licheniformis was constructed and transformed into B. licheniformis B0204, an industrial alpha-amylase producer. The transformants harboring different copies of amyL were developed on kanamycin by using homolog-mediated chromosomal amplification of alpha-amylase gene. The recombinants with different multiple copies of amyL integrated in the chromosome were identified by real-time PCR and evaluated by shake-flask fermentation. Recombinants harboring 2-5 multiple copies of amyL produced more alpha-amylase comparison to the parental strain B0204.
Bacillus ; enzymology ; genetics ; Gene Amplification ; Industrial Microbiology ; Nucleic Acid Amplification Techniques ; Transformation, Genetic ; alpha-Amylases ; biosynthesis ; genetics

OBJECTIVE: Haplotype amplification on germline variants is suggested to imply potential selective advantages and clonal expansion susceptibility and has become an important signature for seeking cancer susceptibility gene.Here we propose an improved association method that fully considers the haplotype amplification status. METHODS: The haplotype amplification status was estimated by the variant allelic frequencies.We adopted a permutation test on variant allelic frequencies to divide the candidate variants into multiple groups.A likelihood clustering method was then applied to establish the neighborhood system of the hidden Markov random field framework.A filtering pipeline was introduced into the proposed method to further refine the candidate variants, including a Wilson's interval filter and a false discovery rate controller.The final candidate set along with the haplotype amplification status was collapsed into the weighted virtual sites for association tests. RESULTS: Through simulated tests on a series of datasets, we compared the type Ⅰ error rates of different minor allele frequencies, which stably fell within 2%, suggesting good robustness of the algorithm.In addition, we compared another 5 published association approaches for Type-Ⅰ and Type-Ⅱ error rates with the proposed method, which resulted in the error rates all within 2%, demonstrating significant advantages and a good statistical ability of the proposed method. CONCLUSIONS The proposed method can accurately identify tumor susceptibility variants in haplotype amplification area with good robustness and stability.
Algorithms ; Cluster Analysis ; Gene Amplification ; Gene Frequency ; Haplotypes ; Humans ; Neoplasms/genetics* ; Polymorphism, Single Nucleotide

BACKGROUND: HLA-B*15 alleles encode molecules belonging to several serologic subtypes, B62, B63, B71, B72, B75, B76, and B77. Using the conventional serologic typing method, assignment of B15 subtypes has often been prone to error specifically in samples exhibiting either an ambiguous or a B15 homozygous reaction pattern. The goal of this study was to establish a supplementary DNA typing method for accurate assignment of B15 subtypes in 'problematic B15 positive samples'. METHODS: B*15 specific gene amplification was performed using a pair of PCR primers that specifically annealed to B*15 and B*46 alleles. Nested PCR was applied to the amplified DNA using 14 sequence specific PCR primer sets. DNA sequencing was used to clarify the assigning of samples exhibiting discrepancies between the results obtained by B*15-specific nested PCR-SSP typing and serology. RESULTS: The B*15-specific nested PCR-SSP typing could clearly discriminate the 9 B*15 alleles expressed in the Korean population. In application of the system to 30 B15 positive serologically typed samples, 4 exhibited discrepancies between serology to PCR-SSP results. DNA sequencing results obtained from the samples were concordant with those from B*15-specific nested PCR-SSP typing. CONCLUSIONS: The established B*15-specific nested PCR-SSP method is superior to serology in accuracy and resolution. Therefore, the method will be useful as a supplementary DNA typing method to clarify HLA-B assignments of 'problematic B15 positive samples' in Koreans.
Alleles ; DNA Fingerprinting* ; DNA* ; Gene Amplification ; HLA-B Antigens ; Polymerase Chain Reaction ; Sequence Analysis, DNA

PURPOSE: The determination of HER-2/neu gene amplification has become necessary for the selection of breast cancer patients to undergo anti-HER-2/neu therapy, using a humanized monoclonal antibody. Chromogenic in situ hybridization (CISH) detection of the HER-2/neu gene, a newly developed method, utilizes a robust and unique-sequence DNA probe labeled with digoxygenin, which is sequentially incubated with antidigoxygenin fluorescein, antifluorescein peroxidase and diaminobenzidine. The aim of this study was to establish a CISH assay for the detection of HER-2/neu amplification. The results were compared with those of the immunohistochemistry (IHC) methods, most frequently used for detecting HER-2/neu alteration. METHODS: CISH was performed in 4 groups of infiltrating breast carcinomas. Each group was comprised of 20 cases in which the HER-2/neu stati had previously been scored on a four value scale: 0, 1+, 2+ and 3+ by IHC. The results of CISH and IHC were compared for each tumor group. The HER-2/neu gene amplification detected by CISH was thpically visualized as large DAB-stained clusters or by many dots in the nucleus. RESULTS: The concordance between the CISH and IHC was 95% (kappa=0.901). Three IHC-positive cases (score 2+) showed no gene amplification and one IHC-negative case (score 1+) showed gene amplification by CISH. CONCLUSION: The current study showed excellent agreement between the CISH and IHC methods. CISH is an accurate, practical and economical approach for determining the HER-2/neu stati in breast carcinomas. It is also a useful methodology for confirming the IHC results in paraffin- embedded tumor samples, so offers a promising alternative to IHC in a routine diagnostic setting.
Breast Neoplasms* ; Breast* ; DNA ; Fluorescein ; Gene Amplification ; Genes, vif ; Humans ; Immunohistochemistry ; In Situ Hybridization* ; Peroxidase

PURPOSE: In breast cancer, Her-2/neu amplification/overexpression predicts a poor clinical outcome, and enhanced survival benefits have been reported with Her-2/neu targeted therapy. Currently, there are several methods for assessing the amplification/overexpression of Her-2/neu, each having advantages and disadvantages. The aim of this work was to establish a reproducible, sensitive and specific method of testing for Her-2/neu, which could be used in diagnostic pathology laboratories. MATERIALS AND METHODS: We compared the immunohistochemistry (IHC) detection of Her-2/neu overexpression, with differential polymerase chain reaction (PCR) to assess the gene amplification of the Her-2/ neu, in 163 cases of invasive ductal carcinoma of the breast using paraffin-embedded tissue. In addition, assessment of the appropriate cut off points was established. RESULTS: The overexpression of the Her-2/neu was detected in 39 (23.9%) cases, and its amplification in 37 (22.7%) cases. The methods were positive in 21.5% of cases and negative in 74.8%. There was a 96.3% concordance between the two methods. The sensitivity and specificity of IHC, compared with PCR, were 94.6 and 96.8%, respectively. CONCLUSION: We conclude that the automation of PCR-based Her-2/neu testing approaches is expected to play an increasing role in the future of Her-2/neu testing. Also, we have demonstrated that IHC is a sensitive and specific method for assessing Her-2/neu stati in breast cancer, compared to PCR. The current study indicates that moderate, or strong, complete membrane staining in> or =10% of tumor cells provides an appropriate cut off point compared with PCR.
Automation ; Breast Neoplasms* ; Breast* ; Carcinoma, Ductal ; Gene Amplification ; Immunohistochemistry* ; Membranes ; Pathology ; Polymerase Chain Reaction* ; Sensitivity and Specificity

PURPOSE: The main clinical significance of HER-2/neu gene amplification is derived from its use as (a) a prognostic indicator, (b) a predictive factor for the sensitivity to chemotherapy, and (c) the identification of cases that are eligible for a specific therapy targeting the HER-2/neu protein. Over- expression of HER-2/neu has been shown to be associated with a poor prognosis for patients with node-positive breast cancer and also possibly for patients with node-negative breast cancer. The purpose of this work was to analyze the HER-2/neu gene amplification and correlate it with other clinico-pathologic parameters. METHODS: The study population consisted of 194 patients with breast cancer who had been treated with curative surgery at the Kangdong Sacred Hospital, Seoul, Korea from 1995 to 2000. Paraffin-embedded tissue samples from the primary tumors were obtained from the hospital archives and the tissue microarray was then constructed. We analyzed the amplification of HER-2/neu gene by the two-color FISH (Fluorescence in situ hybridization) method, and we correlated the results with other clinico-pathologic parameters such as tumor size, stage, histologic grade, lymph node status and hormonal receptor status. RESULTS: For 44 cases (22.7%) of a total 194 cases, the HER-2/neu gene was amplified. HER-2/neu gene amplification was positively correlated with TNM stage and lymph node status, and it was inversely correlated with estrogen receptor positivity. CONCLUSION: For breast cancer, the analysis of the HER-2/neu gene by FISH based on a tissue microarray may be useful.
Breast Neoplasms* ; Breast* ; Drug Therapy ; Estrogens ; Gene Amplification ; Humans ; Korea ; Lymph Nodes ; Prognosis ; Seoul

PURPOSE: C-erbB2/neu gene is potentially amplified and expressed in several tumors. In some cases, this genetic alteration seems to be a prognostic index of both overall survival and time to relapse. We investigated c-erbB2/neu gene amplification and overexpression in bladder cancer and also its relation to tumor grade, stage, and clinical outcome. MATERIALS AND METHODS: We have developed a real-time quantitative PCR assay based on TaqMan fluorescence methodology to evaluate c-erbB2/neu amplification and overexpression and prognostic significance of c-erbB2/neu overexpression in bladder cancer. The study was performed in 20 normal controls and 46 patients with bladder cancer who underwent radical cystectomy or transurethral resection. RESULTS: Twelve cases of bladder cancer (26.1%) showed c-erbB2/neu amplification and 13 cases (28.3%) showed overexpression of c-erbB2/neu. Except one case, a complete correlation between the results of amplification and overexpression of c- erbB2/neu was obtained. The incidence of c-erbB2/neu overexpression was significantly increased among the patients with high grade and/or invasive cancers (p <0.05). A strong association of c-erbB2/neu overexpression with patient survival was also found (p <0.05). CONCLUSIONS: From this study using real-time quantitative PCR, the c-erbB2/neu overexpression, together with tumor grade and stage, may be applicable as the independent prognostic factor for evaluation of invasiveness, metastatic potential and survival of bladder cancer.
Cystectomy ; Fluorescence ; Gene Amplification ; Humans ; Incidence ; Polymerase Chain Reaction* ; Recurrence ; Urinary Bladder Neoplasms* ; Urinary Bladder*