RESUMO
Mutations in the shelterin protein POT1 are associated with chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, angiosarcoma, melanoma, and other cancers. These cancer-associated POT1 (caPOT1) mutations are generally heterozygous, missense, or nonsense mutations occurring throughout the POT1 reading frame. Cancers with caPOT1 mutations have elongated telomeres and show increased genomic instability, but which of the two phenotypes promotes tumorigenesis is unclear. We tested the effects of CAS9-engineered caPOT1 mutations in human embryonic and hematopoietic stem cells (hESCs and HSCs, respectively). HSCs with caPOT1 mutations did not show overt telomere damage. In vitro and in vivo competition experiments showed the caPOT1 mutations did not confer a selective disadvantage. Since DNA damage signaling is known to affect the fitness of HSCs, the data argue that caPOT1 mutations do not cause significant telomere damage. Furthermore, hESC lines with caPOT1 mutations showed no detectable telomere damage response while showing consistent telomere elongation. Thus, caPOT1 mutations are likely selected for during cancer progression because of their ability to elongate telomeres and extend the proliferative capacity of the incipient cancer cells.
Assuntos
Neoplasias/genética , Proteínas de Ligação a Telômeros/genética , Telômero , Animais , Dano ao DNA , Feminino , Humanos , Células K562 , Masculino , Camundongos , Mutação , Complexo Shelterina , Células-TroncoRESUMO
Fibroblast growth factor-23 (FGF23) is a circulating hormone that acts to correct hyperphosphatemic states by inhibiting renal phosphate reabsorption and to prevent hypervitaminosis D by feedback repressing 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) biosynthesis. FGF23 gene expression in the osteoblast/osteocyte is induced by the nuclear vitamin D receptor (VDR) bound to 1,25(OH)2D3, but cycloheximide sensitivity of this induction suggests that it may occur largely via secondary mechanisms requiring cooperating transcription factors. We therefore sought to identify 1,25(OH)2D3-regulated transcription factors that might impact FGF23 expression. Although neither leptin nor interleukin-6 (IL-6) alone affects FGF23 expression, leptin treatment was found to potentiate 1,25(OH)2D3 upregulation of FGF23 in UMR-106 cells, whereas IL-6 treatment blunted this upregulation. Genomic analyses revealed conserved binding sites for STATs (signal transduction mediators of leptin and IL-6 action) along with transcription factor ETS1 in human and other mammalian FGF23 genes. Further, STAT3, STAT1, ETS1, and VDR mRNAs were induced in a dose-dependent manner by 1,25(OH)2D3 in UMR-106 cells. Bioinformatic analysis identified nine potential VDREs in a genomic interval containing human FGF23. Six of the putative VDREs were capable of mediating direct transcriptional activation of a heterologous reporter gene when bound by a 1,25(OH)2D3-liganded VDR complex. A model is proposed wherein 1,25(OH)2D3 upregulates FGF23 production directly via multiple VDREs and indirectly via induction of STAT3, ETS1, and VDR transcription factors that are then activated via cell surface and intracellular signaling to cooperate in the induction of FGF23 through DNA looping and generation of euchromatin architecture.
Assuntos
Osso e Ossos/metabolismo , Calcitriol/farmacologia , Fatores de Crescimento de Fibroblastos/metabolismo , Interleucina-6/farmacologia , Leptina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Células COS , Calcitriol/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Interleucina-6/metabolismo , Leptina/metabolismo , Modelos Animais , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Ratos , Receptores de Calcitriol/metabolismo , Fatores de Transcrição STAT/metabolismoRESUMO
1,25-dihydroxyvitamin D (1,25D), through association with the nuclear vitamin D receptor (VDR), exerts control over a novel endocrine axis consisting of the bone-derived hormone FGF23, and the kidney-expressed klotho, CYP27B1, and CYP24A1 genes, which together prevent hyperphosphatemia/ectopic calcification and govern the levels of 1,25D to maintain bone mineral integrity while promoting optimal function of other vital tissues. When occupied by 1,25D, VDR interacts with RXR to form a heterodimer that binds to VDREs in the region of genes directly controlled by 1,25D (e.g., FGF23, klotho, Npt2c, CYP27B1 and CYP24A1). By recruiting complexes of comodulators, activated VDR initiates a series of events that induces or represses the transcription of genes encoding proteins such as: the osteocyte-derived hormone, FGF23; the renal anti-senescence factor and protein co-receptor for FGF23, klotho; other mediators of phosphate transport including Npt2a/c; and vitamin D hormone metabolic enzymes, CYP27B1 and CYP24A1. The mechanism whereby osteocytes are triggered to release FGF23 is yet to be fully defined, but 1,25D, phosphate, and leptin appear to play major roles. The kidney responds to FGF23 to elicit CYP24A1-catalyzed detoxification of the 1,25D hormone while also repressing both Npt2a/c to mediate phosphate elimination and CYP27B1 to limit de novo 1,25D synthesis. Comprehension of these skeletal and renal actions of 1,25D should facilitate the development of novel mimetics to prevent ectopic calcification, chronic renal and vascular disease, and promote healthful aging.
Assuntos
Receptores de Calcitriol/metabolismo , Animais , Osso e Ossos/metabolismo , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/metabolismo , Humanos , Rim/metabolismo , Proteínas Klotho , Modelos Biológicos , Receptores X de Retinoides/metabolismo , Vitamina D/análogos & derivados , Vitamina D/metabolismoRESUMO
The Bronx was an early epicenter of the COVID-19 pandemic in the USA. We conducted temporal genomic surveillance of 104 SARS-CoV-2 genomes across the Bronx from March October 2020. Although the local structure of SARS-CoV-2 lineages mirrored those of New York City and New York State, temporal sampling revealed a dynamic and changing landscape of SARS-CoV-2 genomic diversity. Mapping the trajectories of mutations, we found that while some became 'endemic' to the Bronx, other, novel mutations rose in prevalence in the late summer/early fall. Geographically resolved genomes enabled us to distinguish between cases of reinfection and persistent infection in two pediatric patients. We propose that limited, targeted, temporal genomic surveillance has clinical and epidemiological utility in managing the ongoing COVID pandemic.
RESUMO
The Bronx was an early epicenter of the COVID-19 pandemic in the USA. We conducted temporal genomic surveillance of SARS-CoV-2 genomes across the Bronx from March-October 2020. Although the local structure of SARS-CoV-2 lineages mirrored those of New York City and New York State, temporal sampling revealed a dynamic and changing landscape of SARS-CoV-2 genomic diversity. Mapping the trajectories of variants, we found that while some became 'endemic' to the Bronx, other, novel variants rose in prevalence in the late summer/early fall. Geographically resolved genomes enabled us to distinguish between cases of reinfection and persistent infection in two pediatric patients. We propose that limited, targeted, temporal genomic surveillance has clinical and epidemiological utility in managing the ongoing COVID pandemic. One sentence summary: Temporally and geographically resolved sequencing of SARS-CoV-2 genotypes enabled surveillance of novel genotypes, identification of endemic viral variants, and clinical inferences, in the first wave of the COVID-19 pandemic in the Bronx.
RESUMO
Isoforms of the mammalian klotho protein serve as membrane co-receptors that regulate renal phosphate and calcium reabsorption. Phosphaturic effects of klotho are mediated in cooperation with fibroblast growth factor receptor-1 and its FGF23 ligand. The vitamin D receptor and its 1,25-dihydroxyvitamin D(3) ligand are also crucial for calcium and phosphate regulation at the kidney and participate in a feedback loop with FGF23 signaling. Herein we characterize vitamin D receptor-mediated regulation of klotho mRNA expression, including the identification of vitamin D responsive elements (VDREs) in the vicinity of both the mouse and human klotho genes. In keeping with other recent studies of vitamin D-regulated genes, multiple VDREs control klotho expression, with the most active elements located at some distance (-31 to -46 kb) from the klotho transcriptional start site. We therefore postulate that the mammalian klotho gene is up-regulated by liganded VDR via multiple remote VDREs. The phosphatemic actions of 1,25-dihydroxyvitamin D(3) are thus opposed via the combined phosphaturic effects of FGF23 and klotho, both of which are upregulated by the liganded vitamin D receptor.
Assuntos
Envelhecimento/metabolismo , Regulação da Expressão Gênica , Glucuronidase/genética , Rim/metabolismo , Receptores de Calcitriol/metabolismo , Elemento de Resposta à Vitamina D , Vitamina D/análogos & derivados , Envelhecimento/efeitos dos fármacos , Animais , Linhagem Celular , Fator de Crescimento de Fibroblastos 23 , Humanos , Proteínas Klotho , Ligantes , Camundongos , RNA Mensageiro/biossíntese , Receptores de Calcitriol/agonistas , Vitamina D/metabolismo , Vitamina D/farmacologiaRESUMO
To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require approximately 70 min to deliver approximately 650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered "hybrid" mechanism of DNA electromigration, in which DNA molecules alternate rapidly between repeating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs.
Assuntos
Eletroforese em Microchip/instrumentação , Eletroforese em Microchip/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/instrumentação , Análise de Sequência de DNA/métodos , DNA/análise , DNA de Cadeia Simples/química , Desenho de Equipamento , Genoma Humano , Humanos , Microscopia de Vídeo/métodos , Polímeros/química , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for "next-gen" sequencing platforms (e.g. the Illumina and 454 machines) - dsDNA molecules within a certain size range are "cut out" of a gel and recovered for subsequent "massively parallel" pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE ( approximately 1989) until now, 20 years later. Fused-silica capillaries and borosilicate glass and plastic microchips quietly offer increasing capacities for fast (and even "ultra-fast"), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro or nanovolume. This Achilles' heel of electrophoresis technologies left an opening through which pooled sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications.
Assuntos
DNA/química , Eletroforese Capilar/métodos , Eletroforese em Microchip/métodos , Acrilamidas/química , Resinas Acrílicas/química , Fenômenos Químicos , DNA de Cadeia Simples/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Processos Estocásticos , ViscosidadeRESUMO
New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (â¼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from â¼35 cells at a time, resulting in â¼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.
Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Processamento de Imagem Assistida por Computador/métodos , Intestinos/citologia , Animais , Big Data , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Dinamina II/metabolismo , Endocitose/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Organoides/citologia , Organoides/diagnóstico por imagem , Organoides/metabolismoRESUMO
True physiological imaging of subcellular dynamics requires studying cells within their parent organisms, where all the environmental cues that drive gene expression, and hence the phenotypes that we actually observe, are present. A complete understanding also requires volumetric imaging of the cell and its surroundings at high spatiotemporal resolution, without inducing undue stress on either. We combined lattice light-sheet microscopy with adaptive optics to achieve, across large multicellular volumes, noninvasive aberration-free imaging of subcellular processes, including endocytosis, organelle remodeling during mitosis, and the migration of axons, immune cells, and metastatic cancer cells in vivo. The technology reveals the phenotypic diversity within cells across different organisms and developmental stages and may offer insights into how cells harness their intrinsic variability to adapt to different physiological environments.
Assuntos
Imageamento Tridimensional/métodos , Microscopia/métodos , Animais , Movimento Celular , Endocitose , Olho/ultraestrutura , Humanos , Mitose , Organelas , Análise de Célula Única , Peixe-ZebraRESUMO
Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Perfilação da Expressão Gênica/métodos , Intestinos/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células Cultivadas , Humanos , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Biomaterial encapsulation of islets has been proposed to improve the long-term success of islet transplantation by recreating a suitable microenvironment and enhancing cell-matrix interactions that affect cellular function. Protein polymer hydrogels previously showed promise as a biocompatible scaffold by maintaining high cell viability. Here, enzymatically-crosslinked protein polymers were used to investigate the effects of varying scaffold properties and of introducing ECM proteins on the viability and function of encapsulated MIN6 ß-cells. Chemical and mechanical properties of the hydrogel were modified by altering the protein concentrations while collagen IV, fibronectin, and laminin were incorporated to reestablish cell-matrix interactions lost during cell isolation. Rheology indicated all hydrogels formed quickly, resulting in robust, elastic hydrogels with Young's moduli similar to soft tissue. All hydrogels tested supported both high MIN6 ß-cell viability and function and have the potential to serve as an encapsulation platform for islet cell delivery in vivo.
Assuntos
Microambiente Celular/fisiologia , Proteínas da Matriz Extracelular/farmacologia , Hidrogéis/metabolismo , Células Secretoras de Insulina/fisiologia , Transplante das Ilhotas Pancreáticas/métodos , Polímeros/farmacologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Materiais Biocompatíveis/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia de Afinidade , Colágeno , Fibronectinas , Células Secretoras de Insulina/efeitos dos fármacos , Laminina , Camundongos , Dados de Sequência Molecular , Reologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Vitamin D is a well-studied agent for cancer chemoprevention and treatment. Its chief circulating metabolite, 25-hydroxyvitamin D, is converted into the active hormone 1,25-dihydroxyvitamin D (1,25D) by the cytochrome P450 enzyme CYP27B1 in kidney and other tissues. 1,25D is then deactivated by CYP24A1 and ultimately catabolized. Colorectal carcinoma cells express CYP27B1 and CYP24A1 that locally regulate 1,25D with potential implications for its impact on carcinogenesis. While 1,25D inhibits cancer growth, the effects of polymorphic variations in genes encoding proteins involved in 1,25D homeostasis are poorly understood. Using an RXR-VDR mammalian two-hybrid (M2H) biologic assay system, we measured vitamin D metabolite uptake and activation of the vitamin D receptor (VDR) pathway in colon cancer cells that expressed one of five CYP27B1 single-nucleotide polymorphisms (SNP) or four CYP24A1 SNPs. Compared with the wild-type control, four of five CYP27B1 SNPs reduced enzymatic activity, whereas one (V166L) increased activity. For CYP24A1, all tested SNPs reduced enzyme activity. Quantitative real-time PCR analyses supported the results of M2H experiments. The observed SNP-directed variation in CYP functionality indicated that vitamin D homeostasis is complex and may be influenced by genetic factors. A comprehensive understanding of 1,25D metabolism may allow for a more personalized approach toward treating vitamin D-related disorders and evaluating risk for carcinogenesis.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Polimorfismo de Nucleotídeo Único , Esteroide Hidroxilases/genética , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/genética , Humanos , Esteroide Hidroxilases/metabolismo , Transcrição Gênica , Vitamina D3 24-HidroxilaseRESUMO
Biomaterials that mimic the extracellular matrix in both modularity and crosslinking chemistry have the potential to recapitulate the instructive signals that ultimately control cell fate. Toward this goal, modular protein polymer-based hydrogels were created through genetic engineering and enzymatic crosslinking. Animal derived tissue transglutaminase (tTG) and recombinant human transglutaminase (hTG) enzymes were used for coupling two classes of protein polymers containing either lysine or glutamine, which have the recognition substrates for enzymatic crosslinking evenly spaced along the protein backbone. Utilizing tTG under physiological conditions, complete crosslinking occurred within 2 min, as determined by particle tracking microrheology. Hydrogel composition impacted the elastic storage modulus of the gel over 4-fold and also influenced microstructure and degree of swelling, but did not appreciably effect degradation by plasmin. Mouse 3T3 and primary human fibroblasts were cultured in both 2- and 3-dimensions without a decrease in cell viability and displayed spreading in 2D. The properties, which are controlled through the specific nature of the protein polymer precursors, render these gels valuable for in situ therapies. Furthermore, the modular hydrogel composition allows tailoring of mechanical and physical properties for specific tissue engineering applications.
Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Polímeros/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Materiais Biocompatíveis/metabolismo , Sobrevivência Celular , Células Cultivadas , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Elasticidade , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Humanos , Hidrogéis/metabolismo , Teste de Materiais , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Células NIH 3T3 , Polímeros/metabolismo , Reologia , Transglutaminases/metabolismo , ViscosidadeRESUMO
The nuclear vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D3 (1,25D), its high affinity renal endocrine ligand, to signal intestinal calcium and phosphate absorption plus bone remodeling, generating a mineralized skeleton free of rickets/osteomalacia with a reduced risk of osteoporotic fractures. 1,25D/VDR signaling regulates the expression of TRPV6, BGP, SPP1, LRP5, RANKL and OPG, while achieving feedback control of mineral ions to prevent age-related ectopic calcification by governing CYP24A1, PTH, FGF23, PHEX, and klotho transcription. Vitamin D also elicits numerous intracrine actions when circulating 25-hydroxyvitamin D3, the metabolite reflecting vitamin D status, is converted to 1,25D locally by extrarenal CYP27B1, and binds VDR to promote immunoregulation, antimicrobial defense, xenobiotic detoxification, anti-inflammatory/anticancer actions and cardiovascular benefits. VDR also affects Wnt signaling through direct interaction with beta-catenin, ligand-dependently blunting beta-catenin mediated transcription in colon cancer cells to attenuate growth, while potentiating beta-catenin signaling via VDR ligand-independent mechanisms in osteoblasts and keratinocytes to function osteogenically and as a pro-hair cycling receptor, respectively. Finally, VDR also drives the mammalian hair cycle in conjunction with the hairless corepressor by repressing SOSTDC1, S100A8/S100A9, and PTHrP. Hair provides a shield against UV-induced skin damage and cancer in terrestrial mammals, illuminating another function of VDR that facilitates healthful aging.
Assuntos
Envelhecimento , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Receptores de Calcitriol/metabolismo , Animais , Cálcio/metabolismo , Fator de Crescimento de Fibroblastos 23 , Humanos , Queratinócitos/citologia , Camundongos , Modelos Biológicos , Osteopontina/metabolismo , Fosfatos/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
By using a microfluidic electrophoresis platform to perform DNA sequencing, genomic information can be obtained more quickly and affordably than the currently employed capillary array electrophoresis instruments. Previous research in our group has shown that physically cross-linked, hydrophobically modified polyacrylamide matrices separate dsDNA more effectively than linear polyacrylamide (LPA) solutions. Expanding upon this work, we have synthesized a series of LPA-co-dihexylacrylamide block copolymers specifically designed to electrophoretically sequence ssDNA quickly and efficiently on a microfluidic device. By incorporating very small amounts of N,N-dihexylacrylamide, a hydrophobic monomer, these copolymer solutions achieved up to approximately 10% increases in average DNA sequencing read length over LPA homopolymer solutions of matched molar mass. Additionally, the inclusion of the small amount of hydrophobe does not significantly increase the polymer solution viscosities, relative to LPA solutions, so that channel loading times between the copolymers and the homopolymers are similar. The resulting polymer solutions are capable of providing enhanced sequencing separations in a short period of time without compromising the ability to rapidly load and unload the matrix from a microfluidic device.
Assuntos
Eletroforese em Microchip/métodos , Análise de Sequência de DNA/métodos , Resinas Acrílicas/síntese química , Resinas Acrílicas/química , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Fluorescência , Reologia , ViscosidadeRESUMO
We have studied the effects of polymer molar mass and concentration on the electrophoretic migration modalities of individual molecules of DNA in LPA, HEC, and PEO solutions via epifluorescent videomicroscopy. While both transient entanglement coupling (TEC) and reptation have been studied in the past, the transition between them has not. Understanding this transition will allow for polymer network properties to be optimized to enhance the speed and resolution of DNA separations in microfluidic devices. Near the overlap threshold concentration, C*, TEC is the dominant observed mode of DNA migration, and the observation frequency of TEC increases with increasing polymer molar mass. As polymer concentration is increased, observed TEC events reduce to zero while DNA reptation events become the only detected mechanism. Individual DNA molecules undergoing both migration mechanisms were counted in solutions of varying polymer molar masses and concentrations and were plotted against a dimensionless polymer concentration, C/C*. The data for LPA reduce to form universal curves with a sharp increase in DNA reptation at approximately 6.5C*. Analogous transition concentrations for PEO and HEC were observed at 5C* and 3.5C*, respectively, reflecting the different physical properties of these polymers. This transition correlates closely with the polymer network entanglement concentration, Ce, as measured by rheological techniques. The electrophoretic mobility of lambda-DNA in LPA polymer solutions was also measured and shows how a balance can be struck between DNA resolution and separation speed by choosing the desired prevalence of DNA reptation.