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Amplicons
Kinds of Amplicons Terms modified by Amplicons Selected AbstractsTesting candidate plant barcode regions in the MyristicaceaeMOLECULAR ECOLOGY RESOURCES, Issue 3 2008S. G. NEWMASTER Abstract The concept and practice of DNA barcoding have been designed as a system to facilitate species identification and recognition. The primary challenge for barcoding plants has been to identify a suitable region on which to focus the effort. The slow relative nucleotide substitution rates of plant mitochondria and the technical issues with the use of nuclear regions have focused attention on several proposed regions in the plastid genome. One of the challenges for barcoding is to discriminate closely related or recently evolved species. The Myristicaceae, or nutmeg family, is an older group within the angiosperms that contains some recently evolved species providing a challenging test for barcoding plants. The goal of this study is to determine the relative utility of six coding (Universal Plastid Amplicon , UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH-psbA) chloroplast loci for barcoding in the genus Compsoneura using both single region and multiregion approaches. Five of the regions we tested were predominantly invariant across species (UPA, rpoB, rpoC1, accD, rbcL). Two of the regions (matK and trnH-psbA) had significant variation and show promise for barcoding in nutmegs. We demonstrate that a two-gene approach utilizing a moderately variable region (matK) and a more variable region (trnH-psbA) provides resolution among all the Compsonuera species we sampled including the recently evolved C. sprucei and C. mexicana. Our classification analyses based on nonmetric multidimensional scaling ordination, suggest that the use of two regions results in a decreased range of intraspecific variation relative to the distribution of interspecific divergence with 95% of the samples correctly identified in a sequence identification analysis. [source] Effect of PCR amplicon size on assessments of clone library microbial diversity and community structureENVIRONMENTAL MICROBIOLOGY, Issue 5 2009Julie A. Huber Summary PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities. [source] Evaluation of a nested PCR test and bacterial culture of swabs from the nasal passages and from abscesses in relation to diagnosis of Streptococcus equi infection (strangles)EQUINE VETERINARY JOURNAL, Issue 1 2006L. MØLLER GRØNBÆK Summary Reasons for performing study: Streptococcus equi is the cause of strangles in horses. To improve diagnostic sensitivity, development and evaluation of DNA-based methods are necessary. Objectives: To evaluate diagnostic methods and observe the pattern of bacterial shedding during natural outbreaks. Methods: Two herds with natural outbreaks of strangles were visited over a period of 15 weeks and 323 samples originating from 35 horses investigated. The diagnostic use of a nested PCR test was evaluated using a collection of 165 isolates of Lancefield group C streptococci (species specificity) and swabs from nasal passages or from abscesses from horses infected with S. equi (diagnostic sensitivity). Results: All 45 S. equi isolates tested positive in the nested PCR, whereas no amplicon was formed when testing the other 120 Lancefield group C isolates. A total of 43 samples were collected from 11 horses showing clinical signs of strangles during the study period. The diagnostic sensitivity for PCR test was 45% and 80% for samples from the nasal passages and abscesses, respectively; the corresponding diagnostic sensitivity for cultivation was 18% and 20%. The diagnostic sensitivity was significantly higher for PCR than for bacterial cultivation. Furthermore, the shedding of S. equi in 2 infected horse populations was evaluated. An intermittent shedding period of S. equi of up to 15 weeks was recorded in this part of the study. It was also shown that shedding of S. equi occurred both from horses with and without clinical signs. Conclusions and potential relevance: The nested PCR test represents a species-specific and -sensitive method for diagnosis of S. equi from clinical samples. It may, however, be desirable in future to develop detection methods with high diagnostic sensitivity and specificity without the potential problems inherent in nested PCR. [source] Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA,DNA hybridizationFEMS MICROBIOLOGY LETTERS, Issue 1 2002Annick Robert-Pillot Abstract We compared the efficiencies of biochemical methods and polymerase chain reaction (PCR) for the identification of Vibrio parahaemolyticus strains. The 122 isolates studied, identified by biochemical tests as V. parahaemolyticus or Vibrio alginolyticus, were tested by R72H PCR assay. The results obtained with the two methods were consistent for 90% of the strains studied. PCR amplification of the R72H fragment generated two unique amplicons, 387 bp and 320 bp in length. For 11% of the strains from seawater, the results of biochemical identification did not correlate with PCR results. DNA,DNA hybridization experiments provided evidence that some strains identified as V. alginolyticus in biochemical tests should be considered members of the V. parahaemolyticus species. We therefore suggest that biochemical tests are not accurate enough for the identification of V. parahaemolyticus isolates and we demonstrate that amplification of the R72H fragment, whether the amplicon is 320 bp or 387 bp long, is a powerful tool for the reliable identification of V. parahaemolyticus. [source] Integrated genomic profiling identifies candidate genes implicated in glioma-genesis and a novel LEO1 - SLC12A1 fusion geneGENES, CHROMOSOMES AND CANCER, Issue 6 2010Linda B. C. Bralten We performed genotyping and exon-level expression profiling on 21 glioblastomas (GBMs) and 19 oligodendrogliomas (ODs) to identify genes involved in glioma initiation and/or progression. Low-copy number amplifications (2.5 < n < 7) and high-copy number amplifications (n > 7) were more frequently observed in GBMs; ODs generally have more heterozygous deletions per tumor. Four high-copy amplicons were identified in more than one sample and resulted in overexpression of the known oncogenes EGFR, MDM2, and CDK4. In the fourth amplicon, RBBP5, a member of the RB pathway, may act as a novel oncogene in GBMs. Not all hCNAs contain known genes, which may suggest that other transcriptional and/or regulatory elements are the target for amplification. Regions with most frequent allelic loss, both in ODs and GBMs, resulted in a reduced expression of known tumor suppressor genes. We identified a homozygous deletion spanning the Pragmin gene in one sample, but direct sequencing of all coding exons in 20 other glioma samples failed to detect additional genetic changes. Finally, we screened for fusion genes by identifying aberrant 5,-3, expression of genes that lie over regions of a copy number change. A fusion gene between exon 11 of LEO1 and exon 10 of SLC12A1 was identified. Our data show that integrated genomic profiling can identify genes involved in tumor initiation, and/or progression and can be used as an approach to identify novel fusion genes. © 2010 Wiley-Liss, Inc. [source] PPFIA1 and CCND1 are frequently coamplified in breast cancerGENES, CHROMOSOMES AND CANCER, Issue 1 2010Ana-Maria Dancau Recently, amplification of PPFIA1, encoding a member of the liprin family located about 600 kb telomeric to CCND1 on chromosome band 11q13, was described in squamous cell carcinoma of head and neck. Because 11q13 amplification is frequent in breast cancer, and PPFIA1 has been suggested to contribute to mammary gland development, we hypothesized that PPFIA1 might also be involved in the 11q13 amplicon in breast cancer and contribute to breast cancer development. A tissue microarray containing more than 2000 human breast cancers was analyzed for gene copy numbers of PPFIA1 and CCND1 by means of fluorescence in situ hybridization. PPFIA1 amplification was found in 248/1583 (15.4%) of breast cancers. Coamplification with CCND1 was found in all (248/248, 100%) PPFIA1 -amplified cancers. CCND1 amplification without PPFIA1 coamplification was found in additional 117 (4.7%) tumors. Amplification of both PPFIA1 and CCND1 were significantly associated with high-grade phenotype (P = 0.0002) but were unrelated to tumor stage (P = 0.7066) or nodal stage (P = 0.5807). No difference in patient prognosis was found between 248 CCND1/PPFIA1 coamplified tumors and 117 tumors with CCND1 amplification alone (P = 0.6419). These data show that PPFIA1 amplification occurs frequently in breast cancer. The higher incidence of CCND1 amplification when compared with PPFIA1, the lack of prognostic relevance of coamplifications, and the fact that PPFIA1 amplification was found exclusively in CCND1 -amplified cancers suggest that PPFIA1 gene copy number changes represent concurrent events of CCND1 amplification rather than specific biological incidents. © 2009 Wiley-Liss, Inc. [source] ERK5 is a target for gene amplification at 17p11 and promotes cell growth in hepatocellular carcinoma by regulating mitotic entryGENES, CHROMOSOMES AND CANCER, Issue 2 2009Keika Zen Using high-density oligonucleotide microarrays, we investigated DNA copy-number aberrations in cell lines derived from hepatocellular carcinomas (HCCs) and detected a novel amplification at 17p11. To identify the target of amplification at 17p11, we defined the extent of the amplicon and examined HCC cell lines for expression of all seven genes in the 750-kb commonly amplified region. Mitogen-activated protein kinase (MAPK) 7, which encodes extracellular-regulated protein kinase (ERK) 5, was overexpressed in cell lines in which the gene was amplified. An increase in MAPK7 copy number was detected in 35 of 66 primary HCC tumors. Downregulation of MAPK7 by small interfering RNA suppressed the growth of SNU449 cells, the HCC cell line with the greatest amplification and overexpression of MAPK7. ERK5, phosphorylated during the G2/M phases of the cell cycle, regulated entry into mitosis in SNU449 cells. In conclusion, our results suggest that MAPK7 is likely the target of 17p11 amplification and that the ERK5 protein product of MAPK7 promotes the growth of HCC cells by regulating mitotic entry. © 2008 Wiley-Liss, Inc. [source] Characterization of amplicons in neuroblastoma: High-resolution mapping using DNA microarrays, relationship with outcome, and identification of overexpressed genesGENES, CHROMOSOMES AND CANCER, Issue 10 2008Anne Fix Somatically acquired chromosomal imbalances are a key feature of neuroblastoma, a heterogeneous pediatric solid tumor. Among these alterations, genomic amplification targeting the MYCN oncogene and observed in about 25,30% of the cases, strongly correlates with advanced stage and poor outcome. In this work, we have used BAC and SNP arrays as well as gene expression arrays to characterize amplifications in neuroblastoma. Eighty-eight distinct BACs defining high-level amplification events were identified in 65 samples, including 43 tumors and 22 cell lines. Although the highest recurrence was observed on chromosome 2, clones on chromosomes 8, 12, 16, and 17 also revealed genomic amplification in several samples. A detailed analysis of the 2p22-2p25 MYCN containing region indicated highly complex patterns in a number of cases. Coamplifications involving MYCN and other regions were explored by FISH in three cell lines. High-resolution arrays then allowed us to further refine the mapping of 25 amplicons in 19 samples, either reducing the size of a single continuous amplicon or increasing the complexity by highlighting multiple noncontiguous regions of amplification. Combined analysis of gene expression profiling and array-CGH data indicated that 12 to 25% of the genes that are targeted by genomic amplification are actually over-expressed in tumor cells, several of them having already been implicated in cancer. Finally, our results suggest that the presence of amplicons localized outside of chromosome 2, in addition to MYCN amplification, may be linked to a particularly severe outcome in neuroblastoma patients. © 2008 Wiley-Liss, Inc. [source] Molecular dissection of the chromosome band 7q21 amplicon in gastroesophageal junction adenocarcinomas identifies cyclin-dependent kinase 6 at both genomic and protein expression levelsGENES, CHROMOSOMES AND CANCER, Issue 8 2008H. van Dekken Amplification of chromosome band 7q21 has been frequently detected in various types of cancer including gastroesophageal junction (GEJ) adenocarcinomas. At present, no gene has been disclosed that can explain this frequent amplification of 7q21 in GEJ carcinomas. Therefore, a detailed genomic analysis of the 7q21 region was performed on a selected series of GEJ adenocarcinomas, i.e., 14 primary adenocarcinomas and 10 cell lines, by array comparative genomic hybridization (aCGH) with a 7q11.22-q31.2 contig array. A distinct peak of amplification was identified at 92.1 Mb in 7q21.2, precisely comprising cyclin-dependent kinase 6 (CDK6), a gene involved in cell cycle regulation. A smaller peak was seen at 116.2 Mb in 7q31.2, the locus of the MET proto-oncogene. No distinct peak was detected for the hepatocyte growth factor (HGF) at 81.3 Mb in 7q21.11. An immunoprofile of HGF, CDK6 and MET revealed a strong correlation between aCGH and immunohistochemical protein expression for CDK6 (P = 0.002). Furthermore, immunohistochemistry did not show expression of CDK6 in Barrett's dysplasia and carcinoma in situ, correlating expression of CDK6 with a malignant phenotype. We conclude that high-resolution genomic analysis and immunoprofiling identify CDK6 as the main candidate target for the recurrent amplification of 7q21 in GEJ adenocarcinomas. © 2008 Wiley-Liss, Inc. [source] Genetic changes in the evolution of multidrug resistance for cultured human ovarian cancer cellsGENES, CHROMOSOMES AND CANCER, Issue 12 2007Timon P. H. Buys The multidrug resistant (MDR) phenotype is often attributed to the activity of ATP-binding cassette (ABC) transporters such as P-glycoprotein (ABCB1). Previous work has suggested that modulation of MDR may not necessarily be a single gene trait. To identify factors that contribute to the emergence of MDR, we undertook integrative genomics analysis of the ovarian carcinoma cell line SKOV3 and a series of MDR derivatives of this line (SKVCRs). As resistance increased, comparative analysis of gene expression showed conspicuous activation of a network of genes in addition to ABCB1. Functional annotation and pathway analysis revealed that many of these genes were associated with the extracellular matrix and had previously been implicated in tumor invasion and cell proliferation. Further investigation by whole genome tiling-path array CGH suggested that changes in gene dosage were key to the activation of several of these overexpressed genes. Remarkably, alignment of whole genome profiles for SKVCR lines revealed the emergence and decline of specific segmental DNA alterations. The most prominent alteration was a novel amplicon residing at 16p13 that encompassed the ABC transporter genes ABCC1 and ABCC6. Loss of this amplicon in highly resistant SKVCR lines coincided with the emergence of a different amplicon at 7q21.12, which harbors ABCB1. Integrative analysis suggests that multiple genes are activated during escalation of drug resistance, including a succession of ABC transporter genes and genes that may act synergistically with ABCB1. These results suggest that evolution of the MDR phenotype is a dynamic, multi-genic process in the genomes of cancer cells. © 2007 Wiley-Liss, Inc. [source] Unscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV -18/MYC ampliconGENES, CHROMOSOMES AND CANCER, Issue 8 2007Chiara Conti Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV -18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2007 Wiley-Liss, Inc. [source] Intrachromosomal amplification of chromosome 21 (iAMP21) may arise from a breakage,fusion,bridge cycleGENES, CHROMOSOMES AND CANCER, Issue 4 2007Hazel M. Robinson Intrachromosomal amplification of chromosome 21 (iAMP21), involving amplification of the RUNX1 gene and duplication of chromosome 21, dup(21q), defines a new cytogenetic subgroup in B-lineage acute lymphoblastic leukemia (ALL) with a poor prognosis. Characterization of this abnormality has become vital to ensure that the most accurate detection method is used. We have previously defined common regions of amplification and deletion of chromosome 21 in these patients, although the level and extent of amplification within the amplicon was highly variable. This study, using interphase fluorescence in situ hybridization (FISH) with chromosome 21 locus specific probes, substantiated these findings in a large series of patients and confirmed that the amplicon always included RUNX1. Thus, FISH with probes directed to the RUNX1 gene remains the most reliable detection method. Metaphase FISH, supported by G- and multiple color chromosomal banding (mBAND) revealed the patient specific morphology and genetic profile of the dup(21q) chromosomes, as well as the complexity of the intrachromosomal changes giving rise to them. These findings suggested that iAMP21 had arisen from a breakage,fusion,bridge cycle: a mechanism previously described in tumors, which we report for the first time in ALL. © 2007 Wiley-Liss, Inc. [source] Altered promoter usage characterizes monoallelic transcription arising with ERBB2 amplification in human breast cancersGENES, CHROMOSOMES AND CANCER, Issue 11 2006Christopher C. Benz Analysis of a collection of human breast cancers (n = 150), enriched in ERBB2-positive cases (n = 57) and involving tumor genotyping relative to population-matched blood genotyping (n = 749) for a common ERBB2 single nucleotide polymorphism Ala(G)1170Pro(C), revealed that ERBB2 amplification in breast cancer is invariably monoallelic. Analysis of paired breast cancer and blood samples from informative (G1170C heterozygotic) ERBB2-positive (n = 12) and ERBB2-negative (n = 17) cases not only confirmed monoallelic amplification and ERBB2 transcriptional overexpression but also revealed that most low ERBB2 expressing breast cancers (12/17) exhibit unbalanced allelic transcription, showing 3-fold to nearly 5,000-fold preferential expression from one of two inherited alleles. To explore cis-acting transcriptional mechanisms potentially selected during ERBB2 amplification, levels of four different ERBB2 transcript variants (5.2, 4.7, 2.1, and 1.4 kb) were correlated with total (4.6 kb) ERBB2 mRNA levels in ERBB2-positive (n = 14) versus ERBB2-negative (n = 43) primary breast cancers. Relative expression of only the 2.1 kb extracellular domain-encoding splice variant and a 4.7 kb mRNA variant that uses an alternative start site were significantly increased in association with ERBB2-positivity, implicating altered promoter usage and selective transcript regulation within the ERBB2 amplicon. Altogether, these findings provide new mechanistic insights into the development of ERBB2-positive breast cancer and strong rationale for delineating candidate cis-acting regulatory elements that may link allele-specific ERBB2 transcription in premalignant breast epithelia with subsequent development of breast cancers bearing monoallelic ERBB2 amplicons. © 2006 Wiley-Liss, Inc. [source] GAB2 is a novel target of 11q amplification in AML/MDSGENES, CHROMOSOMES AND CANCER, Issue 9 2006Andrea Zatkova Chromosome arm 11q amplifications involving the mixed lineage leukemia gene (MLL) locus are rare but recurrent aberrations in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We have recently shown that in addition to the MLL core amplicon, independent sequences in 11q23,24 and/or 11q13.5 are coamplified within the same cytogenetic markers in 90% and 60% of patients, respectively. Here we further narrow down the minimal amplicon in 11q13.5 to 1.17 Mb by means of semi-quantitative PCR and FISH analyses. The newly defined amplicon contains seven genes, including the GRB2 -associated binding protein 2 (GAB2). Using real-time RT-PCR we show a significant transcriptional upregulation of GAB2 in the patients who have GAB2 coamplified with MLL. Thus, the adaptor molecule GAB2 that has already been shown to enhance oncogenic signaling in other neoplasias appears as a novel target of 11q amplification in AML/MDS. © 2006 Wiley-Liss, Inc. [source] Recurrent coamplification of cytoskeleton-associated genes EMS1 and SHANK2 with CCND1 in oral squamous cell carcinomaGENES, CHROMOSOMES AND CANCER, Issue 2 2006Kolja Freier Chromosomal band 11q13 is frequently amplified in oral squamous cell carcinoma (OSCC) and assumed to be critically involved in tumor initiation and progression by proto-oncogene activation. Though cyclin D1 (CCND1) is supposed to be the most relevant oncogene, several additional putative candidate genes are inside this chromosomal region, for which their actual role in tumorigenesis still needs to be elucidated. To characterize the 11q13 amplicon in detail, 40 OSCCs were analyzed by comparative genomic hybridization to DNA microarrays (matrix-CGH) containing BAC clones derived from chromosomal band 11q13. This high-resolution approach revealed a consistent amplicon about 1.7 Mb in size including the CCND1 oncogene. Seven BAC clones covering FGF3, EMS1, and SHANK2 were shown to be frequently coamplified inside the CCND1 amplicon. Subsequent analysis of tissue microarrays by FISH revealed amplification frequencies of 36.8% (88/239) for CCND1, 34.3% (60/175) for FGF3, 37.4% (68/182) for EMS1, and 36.3% (61/168) for SHANK2. Finally, quantitative mRNA expression analysis demonstrated consistent overexpression of CCND1 in all tumors and of EMS1 and SHANK2 in a subset of specimens with 11q13 amplification, but no expression of FGF3 in any of the cases. Our study underlines the critical role of CCND1 in OSCC development and additionally points to the functionally related genes EMS1 and SHANK2, both encoding for cytoskeleton-associated proteins, which are frequently coamplified with CCND1 and therefore could cooperatively contribute to OSCC pathogenesis. © 2005 Wiley-Liss, Inc. [source] Distinct sequences on 11q13.5 and 11q23,24 are frequently coamplified with MLL in complexly organized 11q amplicons in AML/MDS patientsGENES, CHROMOSOMES AND CANCER, Issue 4 2004Andrea Zatkova Amplification within chromosome arm 11q involving the mixed-lineage leukemia gene (MLL) locus is a rare but recurrent aberration in acute myeloid leukemia and myelodysplastic syndrome (AML/MDS). We and others have observed that 11q amplifications in most AML/MDS cases have not been restricted to the chromosomal region surrounding the MLL gene. Therefore, we implemented a strategy to characterize comprehensively 11q amplicons in a series of 13 AML/MDS patients with MLL amplification. Analysis of 4 of the 13 cases by restriction landmark genomic scanning in combination with virtual genome scan and by matrix-based comparative genomic hybridization demonstrated that the 11q amplicon in these four cases consisted of at least three discontinuous sequences derived from different regions of the long arm of chromosome 11. We defined a maximally 700-kb sequence around the MLL gene that was amplified in all cases. Apart from the core MLL amplicon, we detected two additional 11q regions that were coamplified. Using fluorescence in situ hybridization (FISH) analysis, we demonstrated that sequences in 11q13.5 and 11q23,24 were amplified in 8 of 13 and 10 of 12 AML/MDS cases, respectively. Both regions harbor a number of potentially oncogenic genes. In all 13 cases, either one or both of these regions were coamplified with the MLL amplicon. Thus, we demonstrated that 11q amplicons in AML/MDS patients display a complex organization and have provided evidence for coamplification of two additional regions on the long arm of chromosome 11 that may harbor candidate target genes. © 2004 Wiley-Liss, Inc. [source] Genomic imbalances in CML blast crisis: 8q24.12,q24.13 Segment identified as a common region of over-representationGENES, CHROMOSOMES AND CANCER, Issue 4 2003Susan M. Gribble The acute phase of chronic myeloid leukemia (CML) is accompanied by secondary chromosomal changes. The additional changes have a non-random pattern; however, highly abnormal (marker) chromosomes are reported in some 20% of abnormal karyotypes. These marker chromosomes have proved to be beyond the resolution of conventional G-banding analysis. We used molecular cytogenetic techniques to determine the structure of complex chromosome markers in 10 CML-derived cell lines after our investigations of CML patients in blast crisis. Multicolor fluorescence in situ hybridization identified a multitude of structural chromosome aberrations. In addition, genomic gains identified by comparative genomic hybridization (CGH) were mapped to highly complex marker chromosomes in more than one cell line. The most common genomic loss detected by CGH affected chromosome 9, whereas the most common genomic gains affected, in order of frequency, the sequences of 8q, 6, and 13q. The smallest discrete amplification on 8q was identified in cell line MEG-01. This amplicon contains sequences represented by the marker D8S263/RMC08P029 but did not contain the proximal MYC gene or a more distal marker, D8S256/RMC08P025. We determined the size of the amplicon to be less than the chromosome segment 8q24.12,q24.13. The use of region- and locus-specific probes to analyze the organization of highly complex marker structures aided the identification of preferentially amplified genomic regions. The resultant amplifications could harbor gene(s) driving disease progression. © 2003 Wiley-Liss, Inc. [source] Identification of amplified and expressed genes in breast cancer by comparative hybridization onto microarrays of randomly selected cDNA clonesGENES, CHROMOSOMES AND CANCER, Issue 1 2002Jeremy Clark Microarray analysis using sets of known human genes provides a powerful platform for identifying candidate oncogenes involved in DNA amplification events but suffers from the disadvantage that information can be gained only on genes that have been preselected for inclusion on the array. To address this issue, we have performed comparative genome hybridization (CGH) and expression analyses on microarrays of clones, randomly selected from a cDNA library, prepared from a cancer containing the DNA amplicon under investigation. Application of this approach to the BT474 breast carcinoma cell line, which contains amplicons at 20q13, 17q11,21, and 17q22,23, identified 50 amplified and expressed genes, including genes from these regions previously proposed as candidate oncogenes. When considered together with data from microarray expression profiles and Northern analyses, we were able to propose five genes as new candidate oncogenes where amplification in breast cancer cell lines was consistently associated with higher levels of RNA expression. These included the HB01 histone acetyl transferase gene at 17q22,23 and the TRAP100 gene, which encodes a thyroid hormone receptor-associated protein coactivator, at 17q11,21. The results demonstrate the utility of this microarray-based CGH approach in hunting for candidate oncogenes within DNA amplicons. © 2002 Wiley-Liss, Inc. [source] Chromodomain helicase/adenosine triphosphatase DNA binding protein 1,like (CHD1l) gene suppresses the nucleus-to-mitochondria translocation of nur77 to sustain hepatocellular carcinoma cell survival,HEPATOLOGY, Issue 1 2009Leilei Chen Amplification of 1q21 has been detected in 58% to 78% of primary hepatocellular carcinoma cases, suggesting that one or more oncogenes within the amplicon play a critical role in the development of this disease. The chromodomain helicase/adenosine triphosphatase DNA binding protein 1,like gene (CHD1L) is a recently identified oncogene localized at 1q21. Our previous studies have demonstrated that CHD1L has strong tumorigenic ability and confers high susceptibility to spontaneous tumors in a CHD1L -transgenic mouse model. In this study, we demonstrate that the antiapoptotic ability of CHD1L is associated with its interaction with Nur77, a critical member of a p53-independent apoptotic pathway. As the first cellular protein identified to bind Nur77, CHD1L is able to inhibit the nucleus-to-mitochondria translocation of Nur77, which is the key step of Nur77-mediated apoptosis, resulting in the hindrance of the release of cytochrome c and the initiation of apoptosis. Knock-down of CHD1L expression by RNA interference could rescue the mitochondrial targeting of Nur77 and the subsequent apoptosis. Further studies found that the C-terminal Macro domain of CHD1L is responsible for the interaction with Nur77, and a CHD1L mutant lacking residues 600-897 failed to interact with Nur77 and prevented Nur77-mediated apoptosis. More importantly, we found that the inhibition of Nur77-mediated apoptosis by endogenous CHD1L is a critical biological cellular process in hepatocarcinogenesis. Conclusion: We demonstrate in this study that overexpression of CHD1L could sustain tumor cell survival by preventing Nur77-mediated apoptosis. (HEPATOLOGY 2009.) [source] Description and validation of high-throughput simultaneous genotyping and mutation scanning by high-resolution melting curve analysis,HUMAN MUTATION, Issue 6 2009Tú Nguyen-Dumont Abstract Mutation scanning using high-resolution melting curve analysis (HR-melt) is an effective and sensitive method to detect sequence variations. However, the presence of a common SNP within a mutation scanning amplicon may considerably complicate the interpretation of results and increase the number of samples flagged for sequencing by interfering with the clustering of samples according to melting profiles. A protocol describing simultaneous high-resolution gene scanning and genotyping has been reported. Here, we show that it can improve the sensitivity and the efficiency of large-scale case,control mutation screening. Two exons of ATM, both containing an SNP interfering with standard mutation scanning, were selected for screening of 1,356 subjects from an international breast cancer genetics study. Asymmetric PCR was performed in the presence of an SNP-specific unlabeled probe. Stratification of the samples according to their probe-target melting was aided by customized HR-melt software. This approach improved identification of rare known and unknown variants, while dramatically reducing the sequencing effort. It even allowed genotyping of tandem SNPs using a single probe. Hence, HR-melt is a rapid, efficient, and cost-effective tool that can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes.Hum Mutat 30:1,7, 2009. © 2009 Wiley-Liss, Inc. [source] Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling,HUMAN MUTATION, Issue 6 2009Steven F. Dobrowolski Abstract Identifying mitochondrial DNA (mtDNA) sequence variants in human diseases is complicated. Many pathological mutations are heteroplasmic, with the mutant allele represented at highly variable percentages. High-resolution melt (HRM or HRMA) profiling was applied to comprehensive assessment of the mitochondrial genome and targeted assessment of recognized pathological mutations. The assay panel providing comprehensive coverage of the mitochondrial genome utilizes 36 overlapping fragments (301,658,bp) that employ a common PCR protocol. The comprehensive assay identified heteroplasmic mutation in 33 out of 33 patient specimens tested. Allele fraction among the specimens ranged from 1 to 100%. The comprehensive assay panel was also used to assess 125 mtDNA specimens from healthy donors, which identified 431 unique sequence variants. Utilizing the comprehensive mtDNA panel, the mitochondrial genome of a patient specimen may be assessed in less than 1 day using a single 384-well plate or two 96-well plates. Specific assays were used to identify the myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) mutation m.3243A>G, myoclonus epilepsy, ragged red fibers (MERRF) mutation m.8344A>G, and m.1555A>G associated with aminoglycoside hearing loss. These assays employ a calibrated, amplicon-based strategy that is exceedingly simple in design, utilization, and interpretation, yet provides sensitivity to detect variants at and below 10% heteroplasmy. Turnaround time for the genotyping tests is about 1,hr. Hum Mutat 30,1,8, 2009. © 2009 Wiley-Liss, Inc. [source] Ribosomal DNA sequence analysis of mucosa-associated bacteria in Crohn's diseaseINFLAMMATORY BOWEL DISEASES, Issue 6 2004Tom Prindiville MD Abstract Background: Enteric bacteria are implicated in the pathogenesis of Crohn's disease (CD); however, no specific causative organisms have been identified. Aims: This study was undertaken to correlate disease activity with changes in intestinal biota in patients with CD. Subjects: Ribosomal DNA analysis was used to explore the composition of the intestinal biota in patients with (1) CD undergoing colonoscopy, (2) CD undergoing surgical resection, and (3) no inflammatory bowel disease. Methods: Primers targeting bacterial 16S ribosomal DNA (rDNA) were used to amplify bacterial DNA associated with active CD lesions, comparable normal tissue from patients with CD, and normal control tissue. Each amplicon was cloned. Seven hundred thirty-nine rDNA clones were sequenced from 16 biopsies from CD patients, 15 surgical samples, and 10 biopsies from normal control patients. Results: Known extracellular or intracellular pathogens were not found. No rDNA sequence, phylogenetic group, or subgroup was consistently associated with CD lesions compared with normal tissues from the same patients. Colonic biopsies from CD-afflicted patients compared with biopsies from normal control subjects had an increase in facultative bacteria; in small bowel, CD patients had an increase in the Ruminococcus gnavus subgroup with a decrease in the Clostridium leptum and Prevotella nigrescens subgroups. However, differences in small bowel may have reflected individual variation rather than disease association. Surgical samples showed differences when compared with biopsy-derived samples. Conclusions: These findings suggest that CD is not caused by invasive pathogens associated specifically with the sites of lesions but that dysbiosis exists in this condition. [source] Aldehyde oxidase is coamplified with the World's most common Culex mosquito insecticide resistance-associated esterasesINSECT MOLECULAR BIOLOGY, Issue 1 2000J. Hemingway Abstract The evolution and spread of insecticide resistance is an important factor in human disease prevention and crop protection. The mosquito Culex quinquefasciatus is the main vector of the disease filariasis and a member of a species complex which is a common biting nuisance worldwide. The common insecticide resistance mechanism in this species involves germline amplification of the esterases est,21 and est,21. This amplification has arisen once and rapidly spread worldwide. Less common and more variable resistance phenotypes involve coamplification of est,3 and est,1, or individual amplification of a single est,1, different alleles of the same est, and est, gene loci. Est,21 and est,21 are on the same large fragment of amplified DNA (amplicon) 2.7 kb apart. We have now shown that this amplicon contains another full-length gene immediately 5, of est,21 which codes for a molybdenum-containing hydroxylase, with highest homology to aldehyde oxidase (AO) from other organisms. The full-length putative AO gene is not present on the est,3/est,1 or est,1 amplicons, but multiple truncated 5, ends of this gene are present around the presumed est,3/est,1 amplicon breakpoint. Polymerase chain reaction (PCR) analysis of insecticide-susceptible genomic DNA demonstrated that a different allele of the putative AO gene in its non-amplified form is immediately 5, of est,. The ,AO' gene on the est,21/est,21 amplicon is expressed and resistant insects have greater AO activity. This AO activity is sensitive to inhibition by an aldehyde-containing herbicide and pesticide. This enzyme may confer a selective advantage to these insects in the presence of insecticide, as AO in mammals is believed to be important in the detoxification process of several environmental pollutants. [source] Microbial community analysis at crude oil-contaminated soils targeting the 16S ribosomal RNA, xylM, C23O, and bcr genesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009Y. Higashioka Abstract Aims:, The analyses targeting multiple functional genes were performed on the samples of crude oil-contaminated soil, to investigate community structures of organisms involved in monoaromatic hydrocarbon degradation. Methods and Results:, Environmental samples were obtained from two sites that were contaminated with different components of crude oil. The analysis on 16S rRNA gene revealed that bacterial community structures were clearly different between the two sites. The cloning analyses were performed by using primers specific for the catabolic genes involved in the aerobic or anaerobic degradation of monoaromatic hydrocarbons, i.e. xylene monooxygenase (xylM), catechol 2,3-dioxygenase (C23O), and benzoyl-CoA reductase (bcr) genes. From the result of xylM gene, it was suggested that there are lineages specific to the respective sites, reflecting the differences of sampling sites. In the analysis of the C23O gene, the results obtained with two primer sets were distinct from each other. A comparison of these suggested that catabolic types of major bacteria carrying this gene were different between the two sites. As for the bcr gene, no amplicon was obtained from one sample. Phylogenetic analysis revealed that the sequences obtained from the other sample were distinct from the known sequences. Conclusions:, The differences between the two sites were demonstrated in the analyses of all tested genes. As for aerobic cleavage of the aromatic ring, it was also suggested that analysis using two primer sets provide more detailed information about microbial communities in the contaminated site. Significance and Impact of the Study:, The present study demonstrated that analysis targeting multiple functional genes as molecular markers is practical to examine microbial community in crude oil-contaminated environments. [source] Chronic shedding of Campylobacter species in beef cattleJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2004G.D. Inglis Abstract Aims:, To determine the prevalence of chronic shedding of Campylobacter species by beef cattle, a longitudinal study of shedding patterns was conducted in a cohort of 60 beef steers over a 4-month period. Methods and Results:, Steers were maintained in a simulated feedlot setting but individually in pens to minimize transmission among animals. At each collection time, campylobacters in faeces were detected using conventional PCR. In addition, quantities of Campylobacter jejuni and C. lanienae in faeces were measured using real-time quantitative (RTQ) PCR. All of the steers tested shed Campylobacter species during the course of the study, and overall, 90% of the 299 samples tested were positive for Campylobacter DNA. The majority of the animals (86%) shed campylobacters at ,4 sample times. The most prevalent taxon detected in bovine faeces was C. lanienae (56% of samples) followed by C. jejuni (13%), C. hyointestinalis (8%), and C. fetus (2%). No C. coli was detected, and 13% of the faecal samples contained two or more of the above species. Seven (12%) and 34 (57%) animals shed C. jejuni and C. lanienae at ,3 sample times, respectively. For both C. lanienae and C. jejuni, a substantial number of cells were detected in faeces using RTQ-PCR; 27% of the samples positive for C. jejuni contained populations >104 cells g,1 (maximum of 5 × 105 cells g,1), and 44% of samples positive for C. lanienae possessed populations >106 cells g,1 (maximum of 4 × 108 cells g,1). A significant correlation was observed between shedding of C. lanienae and the severity of liver abscesses. In 27% of the samples, an amplicon was obtained for genus-specific but not for the species-specific primers. Sequencing of the partial 16S rRNA gene suggested the presence of at least two undescribed Campylobacter species but this has yet to be confirmed. Conclusions:, A high percentage of feedlot cattle shed large quantities of Campylobacter species in their faeces over a protracted period of time (ca 112 days). Significance and Impact of the Study:, This is the first study of longitudinal shedding patterns of campylobacters in beef cattle using PCR-detection methods. In addition, this is the first use of RTQ-PCR to directly quantify C. jejuni or C. lanienae in faeces. The results of the study show that a large number of cattle (>85%) chronically shed campylobacters in feedlots. [source] Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experienceJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2003C.S. Harrington Abstract Aims: To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP (fla typing) methods previously described for Campylobacter. Methods and Results: The sample set (n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two of the three flaA typing methods amplified flaA alone, whereas one, a multiplex assay, amplified flaB in addition to flaA. DdeI restriction enzyme was employed for all methods, but HinfI was also investigated. 98,100% typeability was obtained for flaA-based methods, but only 93% for the multiplex assay, due to inconsistent amplification of a non-specific product. In addition, there appeared to be selective amplification of flaA over flaB. More DdeI types were generated using a longer flaA PCR amplicon, whilst additional use of HinfI increased the number of types by ca 25%. Inter-laboratory reproducibility for both flaA-based methods was defined at 100%. Conclusions:Fla typing requires standardization with respect to PCR primers and restriction enzymes. This study identified an assay, employing the full flaA gene and DdeI digestion, as an appropriate method on which to standardize. 100% inter-laboratory reproducibility was demonstrated using that method. Significance and Impact of the Study: This work should facilitate progress towards inter-laboratory standardization of fla typing. [source] Occurrence and molecular genotyping of Cryptosporidium spp. in surface waters in Northern IrelandJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2001C.J. Lowery Aims: To investigate the incidence and genotype of Cryptosporidium parvum oocysts in drinking water sources in Northern Ireland for the period 1996,1999, and to compare conventional and molecular methods of detection. Methods and Results: Four hundred and seventy-four waters were investigated by conventional methods, namely immuno-fluorescent antibody detection (IFA; 380) and immuno-magnetic separation-IFA (IMS-IFA; 94), of which 14/474 (3%) were positive. Two hundred and fourteen samples (214/474) were also investigated by PCR techniques, targeting both the 18S rRNA and TRAP-C2 genes, of which 11/214 (5·1%) were positive. These 11 samples were classified as genotype II following sequence analysis of the TRAP-C2 amplicon. Conclusions: This study demonstrated the low incidence of oocysts of C. parvum in water sources in Northern Ireland. Significance and Impact of the Study: Such molecular-based techniques offer a number of advantages over conventional detection methodologies, namely greater sensitivity and specificity as well as the ability to provide accurate genotyping data rapidly, which may be valuable in directing operational management in potential outbreak situations. [source] Prevalence of Actinobacillus actinomycetemcomitans in an ethnic adult Chinese populationJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 9 2001Kai Soo Tan Abstract Aim: The aim of this study was to determine the prevalence and the structure of the leukotoxin promoter region of Actinobacillus actinomycetemcomitans in an ethnic Chinese population. Method: Subgingival plaque samples were collected from 42 patients with moderate to advanced periodontitis and 50 periodontally healthy patients. A. actinomycetemcomitans was detected directly from the crude subgingival plaque by PCR using leukotoxin gene specific primers. The presence of A. actinomycetemcomitans was determined by a single 285 bp PCR amplicon. Results:A. actinomycetemcomitans was found to be present in the subgingival plaque of 68 out of a total of 92 patients examined (74%). 29 out of the 42 periodontitis patients tested were carriers of A. actinomycetemcomitans (69%). Among the periodontally healthy patients studied, 39 out of 50 subjects possessed the bacteria (78%). PCR analysis of the promoter region of the ltx operon revealed that none of the 42 moderate to advanced periodontitis patients examined harboured A.actinomycetemcomitans strains with the JP2-like promoter of the ltx operon, known to enhance leukotoxin expression. 2 out of the 27 advanced periodontitis patients clinically diagnosed as suffering from rapidly progressive periodontitis were found to be carriers of the mildly toxic strain of A. actinomycetemcomitans with the characteristic 652-like promoter. Conclusions: The high prevalence of A. actinomycetemcomitans, regardless of whether the subgingival samples were analysed from patients with healthy or diseased periodontium suggests that this bacterial species is part of the normal oral flora of ethnic Chinese. Our preliminary results also suggested that subjects who harboured the mildly toxic strain of A. actinomycetemcomitans were potentially susceptible to aggressive forms of periodontitis. Zusammenfassung Das Ziel dieser Studie war es, in einer ethnischen Population von Chinesen die Prävalenz von Actinobacillus actinomycetemcomitans und die Struktur der Leukotoxin-Promoterregion zu bestimmen. Von 42 Patienten mit moderater bis fortgeschrittener Parodontitis und 50 parodontal gesunden Patienten wurden subgingivale Plaqueproben entnommen. A. actinomycetemcomitans wurde direkt in der unbehandelten subgingivalen Plaque durch PCR unter Verwendung eines Leukotoxingen-spezifischen Primers nachgewiesen. Das Vorhandensein von A. actinomycetemcomitans wurde mittels eines einzigen 285 bp-PCR-Amplikons bestimmt. Es wurde A. actinomycetemcomitans bei 68 von 92 untersuchten Patienten (74%) vorgefunden. 29 von 42 getesteten Parodontitispatienten waren Träger von A. actinomycetemcomitans (69%). Unter den Studierten parodontal gesunden Patienten besaßen 39 von 50 Personen das Bakterium (78%). Die PCR-Analyse der Promoterregion des ltx -Operons zeigte, dass keiner der 42 untersuchten Patienten mit moderater bis fortgeschrittener Parodontitis den A. actinomycetemcomitans mit dem JP2-ähnlichen Promoter des ltx -Operons, welches die Leukotoxinexpression verstärkt, besaß. Bei 2 der 27 Patienten mit fortgeschrittener Parodontitis wurde klinisch eine rasch fortschreitende Parodontitis diagnostiziert und es wurde der mit geringer Toxizität versehene Stamm des A. actinomycetemcomitans mit dem charakteristischen 652-ähnlichen Promoter vorgefunden. Bedingt durch die hohe Prävalenz von A. actinomycetemcomitans unabhängig davon, ob die Proben von Patienten mit gesundem oder erkranktem Parodontium stammen, lässt sich annehmen, dass diese Bakterienspezies bei ethnischen Chinesen ein Teil normalen Mundflora ist. Unsere vorläufigen Resultate lassen auch annehmen, dass Personen, die den mit geringer Toxizität versehenen Stamm des A. actinomycetemcomitans tragen eine potentielle Anfälligkeit für aggressive Formen der Parodontitis besitzen. Résumé Le but de l'étude présente a été de déterminer la fréquence globale et la structure de la région promoteur de leukotoxine de l'Actinobacillus actinomycetemcomitans (A.a.) dans une population chinoise. Des échantillons de plaque dentaire sous-gingivale ont été prélevés chez 42 patients avec parodontite modérée à avancée et chez 50 patients sains. L'A.a. a été détecté directement dans la plaque sous-gingivale par PCR en utilisant les sites spécifiques de gènes leukotoxines. La présence de l'A.a. a été déterminée par un amplicon PCR de 285 bp. L'A.a. a été décelé dans la plaque sous-gingivale de 68 des 92 patients examinés (74%). 29 des 42 patients avec parodontite ont été reconnus comme porteurs d'A.a. (69%). Parmi les patients sains étudiés, 39 des 50 sujets étaient porteurs de la bactérie (78%). L'analyse PCR de la région promoteur de operon ltx a révélé que des 42 patients avec parodontite modéréà avancée aucun n'avaient de souche A.a. avec le promoteur ressemblant au JP2 de l'operon ltx, reconnu pour acroître la leukotoxine. 2 des 27 patients avec parodontit avancée souffraient d'une parodontite progressant rapidement et étaient porteurs d'une souche moyennement toxique d'A.a. avec la caractéristique du promoteur ressemblant au 652. La fréquence globale importante d'A.a., sans tenir compte si les échantillons sous-gingivaux ont été analysés de patients avec un parodonte sain ou malade, suggère que ces espèces bactériennes font partie de la flore buccale normale de l'ethnie chinoise. Ces résultats indiquent également que les porteurs de la souche peu toxique d'A.a. seraient potentiellement susceptibles à des formes de parodontite agressive. [source] Production and Stability Studies of a Neurotoxin Produced by Clostridium sp.JOURNAL OF FOOD SCIENCE, Issue 3 2006ABSTRACT: A neurotoxigenic Clostridium sp. (RKD) isolated from intestine of decaying fish produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) when tested by mouse protection bioassay. An amplicon of expected size (approximately 700 bp) was generated with primers specific for BoNT/B. Toxin was maximally released in the culture supernatant in the late stationary phase and was dependent on media composition. Growth was optimal in trypticase peptone yeast-extract glucose (TPYG) medium in a pH range of 7.5 to 8.0 and at a temperature between 35°C to 40°C while toxin production was optimum at 37°C (3 to 4 × 103 minimum lethal dose per milliliter) without any protease treatment. There was no correlation between growth and toxin production when cells were grown in media containing different concentrations of NaCl (0% to 5%). Toxin in the culture supernatant was more stable (50% reduction at 50°C in 90 min) than the partially purified fraction. Toxicity was destroyed gradually after increasing the number of freeze-thaw cycles and was almost completely inactivated after 5 cycles. It was completely inactivated by overnight treatment of 1 N NaOH while it retained 1.5% activity with a similar treatment with 1 N HCl. [source] The Application of Miniplex Primer Sets in the Analysis of Degraded DNA from Human Skeletal Remains§JOURNAL OF FORENSIC SCIENCES, Issue 2 2006Kerry L. Opel M.A. ABSTRACT: A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50,280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex® 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex® 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains. [source] | |||||||||||||||||||||||||||||||||||||