Nucleic acid purification using microfabricated silicon structures

A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor. This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300-600%, when the etch depth is varied from 20 to 50 microm. DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. Positive pressure was used to move solutions through the device, removing the need for centrifugation steps. The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 microl of elution buffer. Additionally, the device removed approximately 87% of the protein from a cell lysate. Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor. The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step.     

A tube-cutting device for fractionating preparative gradients from the ultracentrifuge

A device is described which cuts off the bottom of a polyallomer (plastic) centrifuge tube, allowing collection of a preparative gradient from the bottom through a large-diameter opening. The gradient flows downward through a funnel-shaped portal positioned below the tube, and the cross-sectional area of flow is gradually restricted. This device resolved into separate peaks DNA sample components differing in density by only 0.007 g/ml in neutral cesium chloride.     

Comparative evaluation of NANO transport properties for DNA nucleobase based molecular junction devices

The feasibility of electron transport conduction through a guanine base of DNA was investigated and then compared with another component of DNA, i.e., cytosine. A mathematical approach based on the jellium model using non-equilibrium Green’s function combined with semi empirical extended Huckel theory was applied using the Atomistik Tool Kit. This was further used to measure significant transport parameters such as current, conductance, transmission spectra and the HOMO–LUMO gap of the suggested molecular system. An important revelation from our research work is that the cytosine-based molecular device exhibits metallic behavior with current ranging up to 70 μA, and hence establishes itself as a good conductor. On the other hand, the guanine-based device is comparatively less conductive, exhibiting current in the order of 3 μA. Another interesting observation about the guanine-based device is the visibility of a prominent negative differential resistance effect during the positive bias and a tunneling effect during negative bias. The uniform charge transfer through the cytosine device confirms its application as a molecular wire. The observations on the guanine-based device give better insights into its application as a memory device for nano-scale devices.     

A practical device for pinpoint delivery of molecules into multiple neurons in culture

We have developed a device for pinpoint delivery of chemicals, proteins, and nucleic acids into cultured cells. The principle underlying the technique is the flow of molecules from the culture medium into cells through a rupture in the plasma membrane made by a needle puncture. DNA transfection is achieved by stabbing the needle tip into the nucleus. The CellBee device can be attached to any inverted microscope, and molecular delivery can be coupled with conventional live cell imaging. Because the position of the needle relative to the targeted cultured cells is computer-controlled, efficient delivery of molecules such as rhodamine into as many as 100 HeLa cells can be completed in 10 min. Moreover, specific target cells within a single dish can be transfected with multiple DNA constructs by simple changes of culture medium containing different plasmids. In addition, the nano-sized needle tip enables gentle molecular delivery, minimizing cell damage. This method permits DNA transfection into specific hippocampal neurons without disturbing neuronal circuitry established in culture.     

Multifunctional DNA-based biomemory device consisting of ssDNA/Cu heterolayers

In the present study, we developed a novel DNA-based biomemory device that was comprised of ssDNA/Cu heterolayers on Au electrodes. As a conducting material, a thiol-modified single strand DNA (26 bp) was designed and immobilized on the Au electrode without the need for any linker material. Cu(2+) ions, which acted as the active site, were then chemically absorbed on the external structure of ssDNA through electrostatic interactions. The presence of the fabricated ssDNA/Cu heterolayer was confirmed by surface plasmon resonance (SPR) spectroscopy and Raman spectroscopy. Cyclic voltammetry experiments were carried out to investigate the redox properties of ssDNA/Cu hybrids to obtain the oxidation and reduction potential. Based on measured oxidation and reduction potential, a ROM-type, 3-state type, and WORM type DNA memory functions were demonstrated by chronoamperometry (CA) and open circuit potential amperometry (OCPA). This proposed device acts and operates the memory function very well. In the near future, DNA based biomemory device in this study could provide the alternative to the inorganic electronic device when molecular scaled immobilization control and signal measurement are achieved.     

一种简便、高效、经济的从凝胶中回收DNA的方法

目的:尝试一种简便、高效的从凝胶中回收DNA的方法。方法:在Eppendorf管的管底用注射器扎孔,将一小团用Eppendorf管融化后拉成的细丝放入管中,把含有DNA的凝胶放在细丝上,离心,收集从管底流出的液体,经酚氯仿抽提后用乙醇沉淀DNA。结果:经过简单的离心即可近乎100%地回收凝胶中的DNA。结论:使用该方法从琼脂糖凝胶回收DNA,操作简单,回收率高,无其他试剂污染。     

An ON/OFF biosensor based on blockade of ionic current passing through a solid-state nanopore

Single nanopores have attracted interest for their use as biosensing devices. In general, methods involve measuring ionic current blockades associated with translocation of analytes through the nanopore, but the detection of such short time lasting events requires complex equipment and setup that are critical for convenient routine biosensing. Here we present a novel biosensing concept based on a single nanopore in a silicon nitride membrane and two anchor-linked DNA species that forms trans-pore hybrids, realizing a stable blockade of ionic current through the pore. Molecular recognition events affecting the DNA hybrids cause a pore opening and the consequent establishment of an ionic current. In the present implementation of the device, we constructed a magnetic bead/streptavidin/biotin-DNA1/DNA2-biotin/streptavidin/Quantumdot-cluster complex (where DNA1 is a mismatched reverse complement of DNA2) through a sub-micrometric pore and monitored DNA strand displacement events occurring after addition of an oligonucleotide complementary to DNA2. The electric and mechanical aspects of the novel device, as well as its potential in biosensing are discussed.     

A new method of sequencing DNA

An entirely new method of sequencing DNA has been devised that does not use electrophoresis, radioactivity, or fluorescence. The method works by measuring pyrophosphate generated by the DNA polymerization reaction. DNA and DNA polymerase are held by a DEAE-Sepharose column and solutions containing different dNTPs are pumped through. The pyrophosphate generated is measured continuously by a device consisting of a series of columns containing enzymes covalently attached to Sepharose. The alternating copolymer poly(dA.dT) is sequenced as an illustration of the method. Future improvements that will facilitate automation are discussed.     

The adjustable nucleosome: an epigenetic signaling module

This review examines the proposition that the nucleosome, in addition to its role as a DNA packaging device, is a signaling module through which changing environmental and metabolic conditions can influence genomic functions. The role of enzyme-catalyzed post-translational modifications of the core histones is critically assessed, leading to the conclusion that they play varied, often crucial and sometimes causative roles in this signaling process.     

Plastic microfluidic chip for continuous-flow polymerase chain reaction: simulations and experiments

A continuous flow polymerase chain reaction (CF-PCR) device comprises a single fluidic channel that is heated differentially to create spatial temperature variations such that a sample flowing through it experiences the thermal cycling required to induce amplification. This type of device can provide an effective means to detect the presence of a small amount of nucleic acid in very small sample volumes. CF-PCR is attractive for global health applications due to its less stringent requirements for temperature control than for other designs. For mass production of inexpensive CF-PCR devices, fabrication via thermoplastic molding will likely be necessary. Here we study the optimization of a PCR assay in a polymeric CF-PCR device. Three channel designs, with varying residence time ratios for the three PCR steps (denaturation, annealing, and extension), were modeled, built, and tested. A standardized assay was run on the three different chips, and the PCR yields were compared. The temperature gradient profiles of the three designs and the residence times of simulated DNA molecules flowing through each temperature zone were predicted using computational methods. PCR performance predicted by simulation corresponded to experimental results. The effects of DNA template size and cycle time on PCR yield were also studied. The experiments and simulations presented here guided the CF-PCR chip design and provide a model for predicting the performance of new CF-PCR designs prior to actual chip manufacture, resulting in faster turn around time for new device and assay design. Taken together, this framework of combined simulation and experimental development has greatly reduced assay development time for CF-PCR in our lab.     

Detection of DNA recognition events using multi-well field effect devices

We proposed the multi-well field effect device for detection of charged biomolecules and demonstrated the detection principle for DNA recognition events using quasi-static capacitance-voltage (QSCV) measurement. The multi-well field effect device is based on the electrostatic interaction between molecular charges induced by DNA recognition and surface electrons in silicon through the Si(3)N(4)/SiO(2) thin double-layer. Since DNA molecules and DNA binders such as Hoechst 33258 have intrinsic charges in aqueous solutions, respectively, the charge density changes due to DNA recognition events at the Si(3)N(4) surface were directly translated into electrical signal such as a flat band voltage change in the QSCV measurement. The average flat band shifts were 20.7 mV for hybridization and -13.5 mV for binding of Hoechst 33258. From the results of flat band voltage shifts due to hybridization and binding of Hoechst 33258, the immobilization density of oligonucleotide probes at the Si(3)N(4) surface was estimated to be 10(8) cm(-2). The platform based on the multi-well field effect device is suitable for a simple and arrayed detection system for DNA recognition events.     

Dependence of the efficacy of transfecting muscle fibers in vivo on electroporation conditions

This study is a survey of in vivo experiments on transfection of laboratory mouse muscle fibers by electroporation using an original device generating electric impulses. Transfection efficiency proved to depend on DNA dose and the number of electric impulses. It can be increased significantly by electroporation at varying pulse burst polarity. At both direct electrode application to muscles and electroporation through the skin, the muscle fiber transfection was more efficient under electroporation conditions much milder than those usually reported. The use of electroporation method for gene therapy of Duchenne muscular dystrophy is discussed.     

Influence of Electroporation Conditions on Transfection of Muscle Fibers in Vivo

This study is a survey of in vivo experiments on transfection of laboratory mouse muscle fibers by electoporation using an original device generating electric impulses. Transfection efficiency proved to depend on DNA dose and the number of electric impulses. It can be increased significantly by electroporation at varying pulse burst polarity. At both direct electrode application to muscles and electroporation through the skin, the muscle fiber transfection was more efficient under electroporation conditions much milder than those usually reported. The use of electroporation method for gene therapy of Duchenne muscular dystrophy is discussed.     

Structure of bacteriophage SPP1 tail reveals trigger for DNA ejection

The majority of known bacteriophages have long noncontractile tails (Siphoviridae) that serve as a pipeline for genome delivery into the host cytoplasm. The tail extremity distal from the phage head is an adsorption device that recognises the bacterial receptor at the host cell surface. This interaction generates a signal transmitted to the head that leads to DNA release. We have determined structures of the bacteriophage SPP1 tail before and after DNA ejection. The results reveal extensive structural rearrangements in the internal wall of the tail tube. We propose that the adsorption device-receptor interaction triggers a conformational switch that is propagated as a domino-like cascade along the 1600 A-long helical tail structure to reach the head-to-tail connector. This leads to opening of the connector culminating in DNA exit from the head into the host cell through the tail tube.     

一种从琼脂糖凝胶中回收DNA片段的简单电洗脱装置

我们设计了一种简单电洗脱装置,从琼脂糖胶中回收ＤＮＡ。该装置由两个带旋盖的小管、两块透析膜和一个凝胶屏障组成。在电场作用下,ＤＮＡ从凝胶中迁移出来,通过凝胶屏障进入由凝胶屏障和透析膜组成的回收小仓。用微量吸样器收集ＤＮＡ,乙醇沉淀和清洗。该法ＤＮＡ的回收率约８５％;回收的ＤＮＡ可用于基因工程常规实验。     

Highly protective alkalinization by ammonia vapor diffusion in viscosimetric DNA damage assessment

A method for the measurement of viscosities correlated to DNA alterations in alkaline homogenate suspensions is described. The alkaline pH shift to afford cell lysis, DNA unfolding, and denaturation is attained by gaseous ammonia diffusion, thus avoiding shear stress from mechanical mixing. At the same time a stabilizing density gradient is established. This solution is run through a plastic measuring tube that is wide enough to minimize the influence of uneven swelling of the lysing DNA-containing components. Flow times under a carefully controlled water head are registered, and their ratios to control solutions are evaluated. The relative viscosities show a strong and irreversible dependence on shear and on DNase treatment and therefore are considered as essentially DNA derived. The time dependence of the lysate viscosities with and without the DNA-damaging agent bleomycin is given and the dose:activity curves of this agent with sponge homogenates from two orders of Porifera are given with their 50% effective concentration values. The dose:activity curve of an extract from a polluted marine point source is demonstrated. The concentration changes in sponges exposed at differently polluted marine sites are shown. The idea of alkalinization through gaseous diffusion in conjunction with a simple measuring device has already proven a sensitive, reliable, and specific tool in the assessment of DNA damage produced under both laboratory and field conditions.     

Structural and Functional Studies of the Phage Sf6 Terminase Small Subunit Reveal a DNA-Spooling Device Facilitated by Structural Plasticity

In many DNA viruses, genome packaging is initiated by the small subunit of the packaging terminase, which specifically binds to the packaging signal on viral DNA and directs assembly of the terminase holoenzyme. We have experimentally mapped the DNA-interacting region on Shigella virus Sf6 terminase small subunit gp1, which occupies extended surface areas encircling the gp1 octamer, indicating that DNA wraps around gp1 through extensive contacts. High‐resolution structures reveal large-scale motions of the gp1 DNA-binding domain mediated by the curved helix formed by residues 54–81 and an intermolecular salt bridge formed by residues Arg67 and Glu73, indicating remarkable structural plasticity underlying multivalent, pleomorphic gp1:DNA interactions. These results provide spatial restraints for protein:DNA interactions, which enable construction of a three-dimensional pseudo-atomic model for a DNA-packaging initiation complex assembled from the terminase small subunit and the packaging region on viral DNA. Our results suggest that gp1 functions as a DNA-spooling device, which may transform DNA into a specific architecture appropriate for interaction with and cleavage by the terminase large subunit prior to DNA translocation into viral procapsid. This may represent a common mechanism for the initiation step of DNA packaging in tailed double‐stranded DNA bacterial viruses.     

DNA计算机的分子生物学研究进展

DNA(脱氧核糖核酸)计算机研究是一个新领域。从字面上看,它既包含DNA研究也包含计算机的研究,因而也包含DNA技术与计算机技术如何交融的研究。1994年,Adleman在Science上报道了首例DNA计算的研究结果;2001年,Benenson等在Nature报道了一种由DNA分子和相应的酶分子构成的、有图灵机功能的可程序试管型DNA计算机,标志着DNA计算机研究的重大进展。DNA计算机最大的特点是超大规模的并行运算能力和潜在的巨大的数据储存能力。目前DNA计算机研究已涉及许多领域,包括生物学、数学、物理、化学、计算机科学和自动化工程等具体应用,是计算概念上的一次革命。DNA计算机的研究大大促进了DNA分子操作技术尤其是在纳米尺度下操作DNA分子的研究速度。从DNA计算机的基本原理、应用形式、与基因组学研究的重要关系等方面总结和评述了相关研究进展。     

DNA-cleavage studies on N-substituted monocyclic enediynes: enhancement of potency by incorporation of intercalating or electron poor aromatic ring and subsequent design of a novel phototriggerable acyclic enediyne

A number of novel N-substituted enediynes (azaenediynes) 1-4 were synthesized as DNA cleaving agents. Enhancement of DNA cleavage potency was observed with those compounds which could interact with DNA through intercalation of the extended aromatic ring or through electrostatic attraction with electron poor aromatic ring. An acyclic enediyne 5 with a novel phototriggerable device was also synthesized and its DNA-cleaving activity was established.     

Laser Plasma Jet Driven Microparticles for DNA/Drug Delivery

This paper describes a microparticle delivery device that generates a plasma jet through laser ablation of a thin metal foil and uses the jet to accomplish particle delivery into soft living targets for transferring biological agents. Pure gold microparticles of 1 µm size were coated with a plasmid DNA, pIG121Hm, and were deposited as a thin layer on one surface of an aluminum foil. The laser (Nd:YAG, 1064 nm wavelength) ablation of the foil generated a plasma jet that carried the DNA coated particles into the living onion cells. The particles could effectively penetrate the target cells and disseminate the DNA, effecting the transfection of the cells. Generation of the plasma jet on laser ablation of the foil and its role as a carrier of microparticles was visualized using a high-speed video camera, Shimadzu HPV-1, at a frame rate of 500 kfps (2 µs interframe interval) in a shadowgraph optical set-up. The particle speed could be measured from the visualized images, which was about 770 m/s initially, increased to a magnitude of 1320 m/s, and after a quasi-steady state over a distance of 10 mm with an average magnitude of 1100 m/s, started declining, which typically is the trend of a high-speed, pulsed, compressible jet. Aluminum launch pad (for the particles) was used in the present study to make the procedure cost-effective, whereas the guided, biocompatible launch pads made of gold, silver or titanium can be used in the device during the actual clinical operations. The particle delivery device has a potential to have a miniature form and can be an effective, hand-held drug/DNA delivery device for biological applications.