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Results: 1 to 10 of 109

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Human DNA polymerase η accommodates RNA for strand extension.
Su Y, Egli M, Guengerich FP
(2017) J Biol Chem 292: 18044-18051
MeSH Terms: Base Pair Mismatch, DNA Primers, DNA Replication, DNA-Directed DNA Polymerase, Deoxyguanosine, Electrophoretic Mobility Shift Assay, Humans, Kinetics, Nucleic Acid Heteroduplexes, Nucleic Acid Hybridization, Oligodeoxyribonucleotides, Oligoribonucleotides, Pyrimidine Dimers, RNA, Recombinant Proteins, Reverse Transcription, Substrate Specificity, Transcription Elongation, Genetic
Show Abstract · Added March 14, 2018
Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η .
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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18 MeSH Terms
In situ synthesis of peptide nucleic acids in porous silicon for drug delivery and biosensing.
Beavers KR, Mares JW, Swartz CM, Zhao Y, Weiss SM, Duvall CL
(2014) Bioconjug Chem 25: 1192-7
MeSH Terms: Biosensing Techniques, Carcinoma, Hepatocellular, DNA, Drug Delivery Systems, Humans, Liver Neoplasms, Mass Spectrometry, MicroRNAs, Nanoparticles, Nucleic Acid Hybridization, Peptide Nucleic Acids, Porosity, Propylamines, Silanes, Silicon, Tumor Cells, Cultured
Show Abstract · Added April 27, 2017
Peptide nucleic acids (PNA) are a unique class of synthetic molecules that have a peptide backbone and can hybridize with nucleic acids. Here, a versatile method has been developed for the automated, in situ synthesis of PNA from a porous silicon (PSi) substrate for applications in gene therapy and biosensing. Nondestructive optical measurements were performed to monitor single base additions of PNA initiated from (3-aminopropyl)triethoxysilane attached to the surface of PSi films, and mass spectrometry was conducted to verify synthesis of the desired sequence. Comparison of in situ synthesis to postsynthesis surface conjugation of the full PNA molecules showed that surface mediated, in situ PNA synthesis increased loading 8-fold. For therapeutic proof-of-concept, controlled PNA release from PSi films was characterized in phosphate buffered saline, and PSi nanoparticles fabricated from PSi films containing in situ grown PNA complementary to micro-RNA (miR) 122 generated significant anti-miR activity in a Huh7 psiCHECK-miR122 cell line. The applicability of this platform for biosensing was also demonstrated using optical measurements that indicated selective hybridization of complementary DNA target molecules to PNA synthesized in situ on PSi films. These collective data confirm that we have established a novel PNA-PSi platform with broad utility in drug delivery and biosensing.
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16 MeSH Terms
The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry.
Adams NM, Olmsted IR, Haselton FR, Bornhop DJ, Wright DW
(2013) Nucleic Acids Res 41: e103
MeSH Terms: Interferometry, Nucleic Acid Conformation, Nucleic Acid Hybridization, Nucleocapsid Proteins, Oligonucleotide Probes, Oligonucleotides, RNA, RNA Folding, RNA, Viral
Show Abstract · Added March 7, 2014
Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity. These properties suggest that this approach might be useful for detecting biomarkers of infection. In this report, we identify interactions and characteristics of nucleic acid probes that maximize BSI signal upon binding the respiratory syncytial virus nucleocapsid gene RNA biomarker. The number of base pairs formed upon the addition of oligonucleotide probes to a solution containing the viral RNA target correlated with the BSI signal magnitude. Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity. We also demonstrated that locked nucleic acid (LNA) probes improved sensitivity approximately 4-fold compared to DNA probes of the same sequence. We attribute this enhancement in BSI performance to the increased A-form character of the LNA:RNA hybrid. A limit of detection of 624 pM, corresponding to ∼10(5) target molecules, was achieved using nine distinct ∼23-mer DNA probes complementary to regions distributed along the RNA target. Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.
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9 MeSH Terms
Targeted enrichment of specific regions in the human genome by array hybridization.
Igartua C, Turner EH, Ng SB, Hodges E, Hannon GJ, Bhattacharjee A, Rieder MJ, Nickerson DA, Shendure J
(2010) Curr Protoc Hum Genet Chapter 18: Unit 18.3
MeSH Terms: Genome, Human, Humans, Nucleic Acid Hybridization
Show Abstract · Added February 15, 2016
While whole-genome resequencing remains expensive, genomic partitioning provides an affordable means of targeting sequence efforts towards regions of high interest. There are several competitive methods for targeted capture; these include molecular inversion probes, microdroplet-segregated multiplex PCR, and on-array or in-solution capture-by-hybridization. Enrichment of the human exome by array hybridization has been successfully applied to pinpoint the causative allele of Mendelian disorders. This protocol focuses on the application of Agilent 1 M arrays for capture-by-hybridization and sequencing on the Illumina platform, although the library preparation method may be adaptable to other vendors' array platforms and sequencing technologies.
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3 MeSH Terms
Targeted investigation of the Neandertal genome by array-based sequence capture.
Burbano HA, Hodges E, Green RE, Briggs AW, Krause J, Meyer M, Good JM, Maricic T, Johnson PL, Xuan Z, Rooks M, Bhattacharjee A, Brizuela L, Albert FW, de la Rasilla M, Fortea J, Rosas A, Lachmann M, Hannon GJ, Pääbo S
(2010) Science 328: 723-5
MeSH Terms: Amino Acid Substitution, Animals, Fossils, Genes, Genome, Genome, Human, Hominidae, Humans, Nucleic Acid Hybridization, Oligonucleotide Array Sequence Analysis, Pan troglodytes, Proteins, Sequence Alignment, Sequence Analysis, DNA
Show Abstract · Added February 15, 2016
It is now possible to perform whole-genome shotgun sequencing as well as capture of specific genomic regions for extinct organisms. However, targeted resequencing of large parts of nuclear genomes has yet to be demonstrated for ancient DNA. Here we show that hybridization capture on microarrays can successfully recover more than a megabase of target regions from Neandertal DNA even in the presence of approximately 99.8% microbial DNA. Using this approach, we have sequenced approximately 14,000 protein-coding positions inferred to have changed on the human lineage since the last common ancestor shared with chimpanzees. By generating the sequence of one Neandertal and 50 present-day humans at these positions, we have identified 88 amino acid substitutions that have become fixed in humans since our divergence from the Neandertals.
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14 MeSH Terms
Comprehensive genomic analysis reveals clinically relevant molecular distinctions between thymic carcinomas and thymomas.
Girard N, Shen R, Guo T, Zakowski MF, Heguy A, Riely GJ, Huang J, Lau C, Lash AE, Ladanyi M, Viale A, Antonescu CR, Travis WD, Rusch VW, Kris MG, Pao W
(2009) Clin Cancer Res 15: 6790-9
MeSH Terms: Aged, Carcinoma, Cluster Analysis, ErbB Receptors, Female, Genomics, Humans, Immunohistochemistry, Male, Middle Aged, Mutation, Nucleic Acid Hybridization, Oligonucleotide Array Sequence Analysis, Proto-Oncogene Proteins c-kit, Signal Transduction, Thymoma, Thymus Neoplasms
Show Abstract · Added March 24, 2014
PURPOSE - Thymomas and thymic carcinomas are rare intrathoracic malignancies that can be invasive and refractory to conventional treatment. Because these tumors both originate from the thymus, they are often grouped together clinically. However, whether the underlying biology of these tumors warrants such clustering is unclear, and the optimum treatment of either entity is unknown.
EXPERIMENTAL DESIGN - All thymic tumors were profiled for mutations in genes encoding components of the EGFR and KIT signaling pathways, assessed for EGFR and KIT expression by immunohistochemistry, and analyzed by array-based comparative genomic hybridization. Previously untreated tumors were subjected to global gene expression arrays.
RESULTS - We analyzed 45 thymic tumors [thymoma, n = 38 (type A, n = 8; type B2, n = 22; type B3, n = 8); thymic carcinoma, n = 7]. One thymoma and one thymic carcinoma harbored KRAS mutations (G12A and G12V, respectively), and one thymoma had a G13V HRAS mutation. Three tumors displayed strong KIT staining. Two thymic carcinomas harbored somatic KIT mutations (V560del and H697Y). In cell viability assays, the V560del mutant was associated with similar sensitivities to imatinib and sunitinib, whereas the H697Y mutant displayed greater sensitivity to sunitinib. Genomic profiling revealed distinct differences between type A to B2 thymomas versus type B3 and thymic carcinomas. Moreover, array-based comparative genomic hybridization could readily distinguish squamous cell carcinomas of the thymus versus the lung, which can often present a diagnostic challenge.
CONCLUSIONS - Comprehensive genomic analysis suggests that thymic carcinomas are molecularly distinct from thymomas. These data have clinical, pathologic, and therapeutic implications for the treatment of thymic malignancies.
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17 MeSH Terms
High-throughput molecular analysis in lung cancer: insights into biology and potential clinical applications.
Ocak S, Sos ML, Thomas RK, Massion PP
(2009) Eur Respir J 34: 489-506
MeSH Terms: Biomarkers, Chromatin, DNA Methylation, Disease Progression, Epigenesis, Genetic, Gene Expression Profiling, Genomics, Histones, Humans, Lung Neoplasms, Neoplasms, Nucleic Acid Hybridization, Protein Array Analysis, Proteomics, Transcription, Genetic
Show Abstract · Added March 5, 2014
During the last decade, high-throughput technologies including genomic, epigenomic, transcriptomic and proteomic have been applied to further our understanding of the molecular pathogenesis of this heterogeneous disease, and to develop strategies that aim to improve the management of patients with lung cancer. Ultimately, these approaches should lead to sensitive, specific and noninvasive methods for early diagnosis, and facilitate the prediction of response to therapy and outcome, as well as the identification of potential novel therapeutic targets. Genomic studies were the first to move this field forward by providing novel insights into the molecular biology of lung cancer and by generating candidate biomarkers of disease progression. Lung carcinogenesis is driven by genetic and epigenetic alterations that cause aberrant gene function; however, the challenge remains to pinpoint the key regulatory control mechanisms and to distinguish driver from passenger alterations that may have a small but additive effect on cancer development. Epigenetic regulation by DNA methylation and histone modifications modulate chromatin structure and, in turn, either activate or silence gene expression. Proteomic approaches critically complement these molecular studies, as the phenotype of a cancer cell is determined by proteins and cannot be predicted by genomics or transcriptomics alone. The present article focuses on the technological platforms available and some proposed clinical applications. We illustrate herein how the "-omics" have revolutionised our approach to lung cancer biology and hold promise for personalised management of lung cancer.
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15 MeSH Terms
An integrated genomic analysis of lung cancer reveals loss of DUSP4 in EGFR-mutant tumors.
Chitale D, Gong Y, Taylor BS, Broderick S, Brennan C, Somwar R, Golas B, Wang L, Motoi N, Szoke J, Reinersman JM, Major J, Sander C, Seshan VE, Zakowski MF, Rusch V, Pao W, Gerald W, Ladanyi M
(2009) Oncogene 28: 2773-83
MeSH Terms: Adenocarcinoma, Cell Line, Tumor, Cell Proliferation, Chromosome Aberrations, Cluster Analysis, Cyclin-Dependent Kinase Inhibitor p16, Dual-Specificity Phosphatases, ErbB Receptors, Female, Gene Dosage, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Genes, ras, Genome-Wide Association Study, Humans, In Situ Hybridization, Fluorescence, Kaplan-Meier Estimate, Lung Neoplasms, Male, Mitogen-Activated Protein Kinase Phosphatases, Mutation, Nucleic Acid Hybridization, RNA Interference
Show Abstract · Added March 24, 2014
To address the biological heterogeneity of lung cancer, we studied 199 lung adenocarcinomas by integrating genome-wide data on copy number alterations and gene expression with full annotation for major known somatic mutations in this cancer. This showed non-random patterns of copy number alterations significantly linked to EGFR and KRAS mutation status and to distinct clinical outcomes, and led to the discovery of a striking association of EGFR mutations with underexpression of DUSP4, a gene within a broad region of frequent single-copy loss on 8p. DUSP4 is involved in negative feedback control of EGFR signaling, and we provide functional validation for its role as a growth suppressor in EGFR-mutant lung adenocarcinoma. DUSP4 loss also associates with p16/CDKN2A deletion and defines a distinct clinical subset of lung cancer patients. Another novel observation is that of a reciprocal relationship between EGFR and LKB1 mutations. These results highlight the power of integrated genomics to identify candidate driver genes within recurrent broad regions of copy number alteration and to delineate distinct oncogenetic pathways in genetically complex common epithelial cancers.
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23 MeSH Terms
Hybrid selection of discrete genomic intervals on custom-designed microarrays for massively parallel sequencing.
Hodges E, Rooks M, Xuan Z, Bhattacharjee A, Benjamin Gordon D, Brizuela L, Richard McCombie W, Hannon GJ
(2009) Nat Protoc 4: 960-74
MeSH Terms: Animals, Base Sequence, DNA Primers, Gene Library, Genomics, Humans, Mice, Nucleic Acid Hybridization, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction, Sensitivity and Specificity, Sequence Analysis, DNA
Show Abstract · Added February 15, 2016
Complementary techniques that deepen information content and minimize reagent costs are required to realize the full potential of massively parallel sequencing. Here, we describe a resequencing approach that directs focus to genomic regions of high interest by combining hybridization-based purification of multi-megabase regions with sequencing on the Illumina Genome Analyzer (GA). The capture matrix is created by a microarray on which probes can be programmed as desired to target any non-repeat portion of the genome, while the method requires only a basic familiarity with microarray hybridization. We present a detailed protocol suitable for 1-2 microg of input genomic DNA and highlight key design tips in which high specificity (>65% of reads stem from enriched exons) and high sensitivity (98% targeted base pair coverage) can be achieved. We have successfully applied this to the enrichment of coding regions, in both human and mouse, ranging from 0.5 to 4 Mb in length. From genomic DNA library production to base-called sequences, this procedure takes approximately 9-10 d inclusive of array captures and one Illumina flow cell run.
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12 MeSH Terms
Molecular beacons can assess changes in expression and 3'-polyadenylation of human eNOS mRNA.
Jones R, Baker MB, Weber M, Harrison DG, Bao G, Searles CD
(2009) Am J Physiol Cell Physiol 296: C498-504
MeSH Terms: Animals, Cells, Cultured, Endothelial Cells, Fluorescence Resonance Energy Transfer, Fluorescent Dyes, Gene Expression Regulation, Enzymologic, Humans, Hydroxymethylglutaryl-CoA Reductase Inhibitors, Lovastatin, Lung, Mice, Nitric Oxide Synthase Type III, Nucleic Acid Hybridization, Oligonucleotide Probes, Physical Exertion, Polyadenylation, Polymerase Chain Reaction, RNA Interference, RNA, Messenger, Reproducibility of Results, Stress, Mechanical
Show Abstract · Added December 10, 2013
The endothelium plays an essential role in maintaining vascular homeostasis, and it fulfills this role by modulating intracellular signaling and gene expression in response to chemical and mechanical stimuli. Assessing changes in endothelial gene expression is essential to understanding how physiological and pathophysiological processes modulate vascular homeostasis. Here we describe the use of molecular beacons to rapidly and quantitatively assess expression and 3'-polyadenylation of a gene that is important for vascular homeostasis, endothelial nitric oxide synthase (eNOS). Single- and dual-fluorescence resonance energy transfer (FRET) molecular beacon hybridization assays were developed to measure changes in mRNA levels and 3'-polyadenylation, respectively, in primary human endothelial cell cultures subjected to laminar shear stress or statin treatment. Optimized beacon hybridization assays took approximately 15 min to perform, and eNOS mRNA levels were validated by quantitative real-time RT-PCR. Competitive inhibition assays and posttranscriptional silencing of eNOS expression were used to verify the specificity of molecular beacon fluorescence. Finally, the dual-FRET method was used to assess eNOS polyadenylation in tissues isolated from mice subjected to exercise training. These data demonstrate that molecular beacons can be used to rapidly and efficiently measure endothelial gene expression and 3'-polyadenylation. This approach could easily be adapted for studies of other endothelial genes and has promise for applications in live endothelial cells.
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21 MeSH Terms