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

The author detected the genetic diversity and genetic relationship within and among eight medicinal species of Dipsacus by the approach of sequence-related amplified polymorphism (SRAP). The associated genetic parameters were calculated by POPGENE 1.31. The Genetic distance was calculated by TREECONW and the systematic diagrams of genetic relationship were clustered by UPG-MA. The results showed that, using 26 primers, a total of 558 bands were produced, of which 539 were polymorphic loci. There was a high level of genetic diversity among species (PPB = 96.59%, Na = 1.9659, Na = 1.3375, H = 0.2143, I = 0.3423). However, genetic diversity was lower within species, the average of genetic parameters was PPB = 6.97%, Na = 1.0697, Na = 1.0311, H = 0.0187, I = 0.0291. The Nei's genetic differentiation coefficient was 0.9126, indicated that most of the genetic variation existed among species. By clustering analysis, different individuals gathered in the same group and the classified result of SRAP marker between traditional modal characters was almost same. The results confirmed that SRAP marker can be used as one of the effective methods to reveal the genetic diversity and relationship among medicinal species of Dipsacus.
China ; Dipsacaceae ; classification ; genetics ; Gene Amplification ; Genetic Variation ; Plants, Medicinal ; classification ; genetics ; Polymorphism, Genetic

OBJECTIVETo prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).

METHODSThe CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA.

RESULTSCFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability.

CONCLUSIONThe reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.

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*

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