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https://www.readbyqxmd.com/read/28213308/simultaneous-estimation-of-detection-sensitivity-and-absolute-copy-number-from-digital-pcr-serial-dilution
#1
Xutao Deng, Brian S Custer, Michael P Busch, Sonia Bakkour, Tzong-Hae Lee
Digital polymerase chain reaction (dPCR) is a refinement of the conventional PCR approach to nucleic acid detection and absolute quantification. Digital PCR works by partitioning a sample of DNA or cDNA into many individual, parallel PCR reactions. Current quantification methods rely on the assumption that the PCR reactions are always able to detect single target molecules. When the assumption does not hold, the copy numbers will be severely underestimated. We developed a novel dPCR quantification method which determines whether the single copy assumption is violated or not by simultaneously estimating the assay sensitivity and the copy numbers using serial dilution data sets...
February 1, 2017: Computational Biology and Chemistry
https://www.readbyqxmd.com/read/28211674/monte-carlo-modeling-based-digital-loop-mediated-isothermal-amplification-on-a-spiral-chip-for-absolute-quantification-of-nucleic-acids
#2
Yun Xia, Shuangqian Yan, Xian Zhang, Peng Ma, Wei Du, Xiaojun Feng, Bi-Feng Liu
Digital loop-mediated isothermal amplification (dLAMP) is an attractive approach for absolute quantification of nucleic acids with high sensitivity and selectivity. Theoretical and numerical analysis of dLAMP provides necessary guidance for the design and analysis of dLAMP devices. In this work, a mathematical model was proposed based on Monte Carlo method and the theories of Poisson statistics and chemometrics. To examine the established model, we fabricated a spiral chip with 1200 uniform and discrete reaction chambers (9...
February 17, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28193077/partition-volume-variability-in-digital-polymerase-chain-reaction-methods-polydispersity-causes-bias-but-can-improve-precision
#3
Joel Tellinghuisen
The role of partition volume variability, or polydispersity, in digital polymerase chain reaction methods is examined through formal considerations and Monte Carlo simulations. Contrary to intuition, polydispersity causes little precision loss for low average copy number per partition μ and can actually improve precision when μ exceeds ∼4. It does this by negatively biasing the estimates of μ, thus increasing the number of negative (null) partitions N0. In keeping with binomial statistics, this increases the relative precision of N0 and hence of the biased estimate m of μ...
December 20, 2016: Analytical Chemistry
https://www.readbyqxmd.com/read/28193036/international-comparison-of-enumeration-based-quantification-of-dna-copy-concentration-using-flow-cytometric-counting-and-digital-polymerase-chain-reaction
#4
Hee-Bong Yoo, Sang-Ryoul Park, Lianhua Dong, Jing Wang, Zhiwei Sui, Jernej Pavšič, Mojca Milavec, Muslum Akgoz, Erkan Mozioğlu, Philippe Corbisier, Mátrai Janka, Bruno Cosme, Janaina J de V Cavalcante, Roberto Becht Flatshart, Daniel Burke, Michael Forbes-Smith, Jacob McLaughlin, Kerry Emslie, Alexandra S Whale, Jim F Huggett, Helen Parkes, Margaret C Kline, Jo Lynne Harenza, Peter M Vallone
Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter...
December 20, 2016: Analytical Chemistry
https://www.readbyqxmd.com/read/28191267/single-cell-digital-polymerase-chain-reaction-on-self-priming-compartmentalization-chip
#5
Qiangyuan Zhu, Lin Qiu, Yanan Xu, Guang Li, Ying Mu
Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell...
January 2017: Biomicrofluidics
https://www.readbyqxmd.com/read/28164427/application-of-circulating-tumor-dna-in-prospective-clinical-oncology-trials-standardization-of-pre-analytical-conditions
#6
Lisanne F van Dessel, Nick Beije, Jean C A Helmijr, Silvia R Vitale, Jaco Kraan, Maxime P Look, Ronald de Wit, Stefan Sleijfer, Maurice P H M Jansen, John W M Martens, Martijn P J K Lolkema
Circulating tumor DNA (ctDNA) has emerged as a potential new biomarker with diagnostic, predictive and prognostic applications for various solid tumor types. Before beginning large prospective clinical trials to prove the added value of utilizing ctDNA in clinical practice, it is essential to investigate the effects of various pre-analytical conditions on the quality of cell-free DNA (cfDNA) in general and of ctDNA in particular in order to optimize and standardize these conditions. Whole blood samples were collected from patients with metastatic cancer bearing a known somatic variant...
February 6, 2017: Molecular Oncology
https://www.readbyqxmd.com/read/28154880/accurate-quantitation-of-circulating-cell-free-mitochondrial-dna-in-plasma-by-droplet-digital-pcr
#7
Wei Ye, Xiaojun Tang, Chu Liu, Chaowei Wen, Wei Li, Jianxin Lyu
To establish a method for accurate quantitation of circulating cell-free mitochondrial DNA (ccf-mtDNA) in plasma by droplet digital PCR (ddPCR), we designed a ddPCR method to determine the copy number of ccf-mtDNA by amplifying mitochondrial ND1 (MT-ND1). To evaluate the sensitivity and specificity of the method, a recombinant pMD18-T plasmid containing MT-ND1 sequences and mtDNA-deleted (ρ(0)) HeLa cells were used, respectively. Subsequently, different plasma samples were prepared for ddPCR to evaluate the feasibility of detecting plasma ccf-mtDNA...
February 2, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28152506/detection-fidelity-of-ar-mutations-in-plasma-derived-cell-free-dna
#8
Alexa Goldstein, Patricia Valda Toro, Justin Lee, John L Silberstein, Mary Nakazawa, Ian Waters, Karen Cravero, David Chu, Rory L Cochran, Minsoo Kim, Daniel Shinn, Samantha Torquato, Robert M Hughes, Aparna Pallavajjala, Michael A Carducci, Channing J Paller, Samuel R Denmeade, Bruce Kressel, Bruce J Trock, Mario A Eisenberger, Emmanuel S Antonarakis, Ben H Park, Paula J Hurley
Somatic genetic alterations including copy number and point mutations in the androgen receptor (AR) are associated with resistance to therapies targeting the androgen/AR axis in patients with metastatic castration resistant prostate cancer (mCRPC). Due to limitations associated with biopsying metastatic lesions, plasma derived cell-free DNA (cfDNA) is increasingly being used as substrate for genetic testing. AR mutations detected by deep next generation sequencing (NGS) of cfDNA from patients with mCRPC have been reported at allelic fractions ranging from over 25% to below 1%...
January 31, 2017: Oncotarget
https://www.readbyqxmd.com/read/28147232/use-of-ubiquitous-highly-heterozygous-copy-number-variants-and-digital-droplet-pcr-to-monitor-chimerism-after-allogeneic-haematopoietic-stem-cell-transplantation
#9
John B Whitlam, Ling Ling, Michael Swain, Tom Harrington, Oksana Mirochnik, Ian Brooks, Sara Cronin, Jackie Challis, Vida Petrovic, Damien L Bruno, Francoise Mechinaud, Rachel Conyers, Howard Slater
Chimerism analysis has an important role in the management of allogeneic haematopoietic stem cell transplantation. It informs response to disease relapse, graft rejection and graft-versus-host disease. We have developed a method for chimerism analysis using ubiquitous copy number variation (CNV), which has the benefit of a "negative background" against which multiple independent informative markers are absolutely quantified using digital droplet PCR. A panel of up to 38 CNV markers with homozygous deletion frequencies of approximately 0...
January 29, 2017: Experimental Hematology
https://www.readbyqxmd.com/read/28145101/typing-and-copy-number-determination-for-hla-drb3-drb4-and-drb5-from-next-generation-sequencing-data
#10
Y Zhang, Y Song, H Cao, X Mo, H Yang, J Wang, Z Lu, T Zhang
BACKGROUND: HLA-DRB3, DRB4 and DRB5 (DRB3/4/5) are paralogues of HLA-DRB1. They have important roles in transplantation and have been reported to be related to many diseases. HLA typing methods for DRB3/4/5 based on NGS data have many limitations now, such as need of polymerase chain reaction (PCR) or low accuracy. MATERIALS AND METHODS: We present a HLA typing method for DRB3/4/5 based on read mapping and haplotype assembly from NGS data. Also, copy number of DRB3/4/5 is determined by a k-means clustering method according to ratio of sequencing depth between DRB3/4/5 and DRB1...
February 1, 2017: HLA
https://www.readbyqxmd.com/read/28145097/multiregion-ultra-deep-sequencing-reveals-early-intermixing-and-variable-levels-of-intratumoral-heterogeneity-in-colorectal-cancer
#11
Yuka Suzuki, Sarah Boonhsi Ng, Clarinda Chua, Wei Qiang Leow, Jermain Chng, Shi Yang Liu, Kalpana Ramnarayanan, Anna Gan, Dan Liang Ho, Rachel Ten, Yan Su, Alexandar Lezhava, Jiunn Herng Lai, Dennis Koh, Kiat Hon Lim, Patrick Tan, Steven G Rozen, Iain Beehuat Tan
Intratumor heterogeneity (ITH) contributes to cancer progression and chemoresistance. We sought to comprehensively describe ITH of somatic mutations, copy number, and transcriptomic alterations involving clinically and biologically relevant gene pathways in colorectal cancer (CRC). We performed multiregion, high-depth (384× on average) sequencing of 799 cancer-associated genes in 24 spatially separated primary tumor and nonmalignant tissues from four treatment-naïve CRC patients. We then used ultra-deep sequencing (17 075× on average) to accurately verify the presence or absence of identified somatic mutations in each sector...
February 2017: Molecular Oncology
https://www.readbyqxmd.com/read/28138851/discovery-of-rare-haplotypes-by-typing-millions-of-single-molecules-with-bead-emulsion-haplotyping-beh
#12
Elisabeth Palzenberger, Ronja Reinhardt, Leila Muresan, Barbara Palaoro, Irene Tiemann-Boege
Characterizing polymorphisms on single molecules renders the phase of different alleles, and thus, haplotype information. Here, we describe a high-throughput method to genotype hundreds-of thousands single molecules in parallel using bead-emulsion haplotyping (BEH). Haplotyping via BEH is an emulsion-PCR-based method that was adapted to amplify multiple DNA fragments on paramagnetic, microscopic beads within a compartment formed by an aqueous-oil emulsion. This generates beads covered by thousands of clonal copies from several polymorphic regions of an initial DNA molecule that are then genotyped with fluorescently labeled probes...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28125683/differences-in-amy1-gene-copy-numbers-derived-from-blood-buccal-cells-and-saliva-using-quantitative-and-droplet-digital-pcr-methods-flagging-the-pitfall
#13
Delicia Shu Qin Ooi, Verena Ming Hui Tan, Siong Gim Ong, Yiong Huak Chan, Chew Kiat Heng, Yung Seng Lee
INTRODUCTION: The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. METHODS: Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant...
2017: PloS One
https://www.readbyqxmd.com/read/28124757/inter-laboratory-assessment-of-different-digital-pcr-platforms-for-quantification-of-human-cytomegalovirus-dna
#14
Jernej Pavšič, Alison Devonshire, Andrej Blejec, Carole A Foy, Fran Van Heuverswyn, Gerwyn M Jones, Heinz Schimmel, Jana Žel, Jim F Huggett, Nicholas Redshaw, Maria Karczmarczyk, Erkan Mozioğlu, Sema Akyürek, Müslüm Akgöz, Mojca Milavec
Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials...
January 26, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28113430/piecewise-mapping-in-hevc-lossless-intra-prediction-coding
#15
Victor Sanchez, Francesc Auli-Llinas, Joan Serra-Sagrista
The lossless intra-prediction coding modality of the High Efficiency Video Coding (HEVC) standard provides high coding performance while allowing frame-by-frame basis access to the coded data. This is of interest in many professional applications such as medical imaging, automotive vision and digital preservation in libraries and archives. Various improvements to lossless intra-prediction coding have been proposed recently, most of them based on sample-wise prediction using Differential Pulse Code Modulation (DPCM)...
May 19, 2016: IEEE Transactions on Image Processing: a Publication of the IEEE Signal Processing Society
https://www.readbyqxmd.com/read/28100494/quantification-of-bk-virus-standards-by-quantitative-real-time-pcr-and-droplet-digital-pcr-is-confounded-by-multiple-virus-populations-in-the-who-bkv-international-standard
#16
Allen C Bateman, Alexander L Greninger, Ederlyn E Atienza, Ajit P Limaye, Keith R Jerome, Linda Cook
BACKGROUND: The WHO recently released a BK virus (BKV) international standard. This study evaluated the WHO international standard and commercially available BKV standards by quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). METHODS: WHO, Exact Diagnostics, Acrometrix, and Zeptometrix BKV standards were tested by qPCR and ddPCR. Two preparations of NIST BKV clones were also tested. Nucleic acid was extracted with the Roche MP96 and MPLC, followed by quantification in duplicate...
January 18, 2017: Clinical Chemistry
https://www.readbyqxmd.com/read/28089353/cerebrospinal-fluid-mitochondrial-dna-in-the-alzheimer-s-disease-continuum
#17
Laura Cervera-Carles, Daniel Alcolea, Ainara Estanga, Mirian Ecay-Torres, Andrea Izagirre, Montserrat Clerigué, Maite García-Sebastián, Jorge Villanúa, Clàudia Escalas, Rafael Blesa, Pablo Martínez-Lage, Alberto Lleó, Juan Fortea, Jordi Clarimón
Low levels of cell-free mitochondrial DNA (mtDNA) in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients have been identified and proposed as a novel biomarker for the disease. The lack of validation studies of previous results prompted us to replicate this finding in a comprehensive series of patients and controls. We applied droplet digital polymerase chain reaction in CSF specimens from 124 patients representing the AD spectrum and 140 neurologically healthy controls. The following preanalytical and analytical parameters were evaluated: the effect of freeze-thaw cycles on mtDNA, the linearity of mtDNA load across serial dilutions, and the mtDNA levels in the diagnostic groups...
December 22, 2016: Neurobiology of Aging
https://www.readbyqxmd.com/read/28077562/sensitive-and-inexpensive-digital-dna-analysis-by-microfluidic-enrichment-of-rolling-circle-amplified-single-molecules
#18
Malte Kühnemund, Iván Hernández-Neuta, Mohd Istiaq Sharif, Matteo Cornaglia, Martin A M Gijs, Mats Nilsson
Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1...
January 10, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28061740/prevalence-and-determinants-of-bullying-among-health-care-workers-in-portugal
#19
Pedro Norton, Viviana Costa, Joel Teixeira, Ana Azevedo, António Roma-Torres, Joana Amaro, Liliana Cunha
Bullying is defined as systematic exposure to humiliation as well as hostile and violent behaviors against one or more individuals. These behaviors are a serious, growing problem, which affects a significant proportion of health care professionals. To support the hospital's risk management policy, a cross-sectional study was undertaken to determine the prevalence of bullying in this institution and identify the determinants of bullying. Bullying was measured using the Negative Acts Questionnaire-Revised, Portuguese version (NAQ-R), a self-administered tool...
January 1, 2017: Workplace Health & Safety
https://www.readbyqxmd.com/read/28060259/measurement-of-differentially-methylated-ins-dna-species-in-human-serum-samples-as-a-biomarker-of-islet-%C3%AE-cell-death
#20
Sarah A Tersey, Jennifer B Nelson, Marisa M Fisher, Raghavendra G Mirmira
The death of islet β cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank hyperglycemia, especially in type 1 diabetes. The development of sensitive and reliable biomarkers of β cell death may allow for early therapeutic intervention to prevent or delay the development of diabetes. Recently, several groups including our own have reported that cell-free, differentially methylated DNA encoding preproinsulin (INS) in the circulation is correlated to β cell death in pre-type 1 diabetes and new-onset type 1 diabetes...
December 21, 2016: Journal of Visualized Experiments: JoVE
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