DNA nanotechnology and fluorescence applications

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Functional DNA nanotechnology: emerging applications of DNAzymes and aptamers

In the past 25 years, DNA molecules have been utilized both as powerful synthetic building blocks to create nanoscale architectures and as versatile programmable templates for assembly of nanomaterials. In parallel, the functions of DNA molecules have been expanded from pure genetic information storage to catalytic functions like those of protein enzymes (DNAzymes) and specific binding functions like antibodies (aptamers). In the past few years, a new interdisciplinary field has emerged that aims to combine functional DNA biology with nanotechnology to generate more dynamic and controllable DNA-based nanostructures or DNA-templated nanomaterials that are responsive to chemical stimuli.     

Oligonucleotide-based microarray for DNA methylation analysis: principles and applications

Gene silencing via promoter CpG island hypermethylation offers tumor cells growth advantages. This epigenetic event is pharmacologically reversible, and uncovering a unique set of methylation-silenced genes in tumor cells can bring a new avenue to cancer treatment. However, high-throughput tools capable of surveying the methylation status of multiple gene promoters are needed for this discovery process. Herein we describe an oligonucleotide-based microarray technique that is both versatile and sensitive in revealing hypermethylation in defined regions of the genome. DNA samples are bisulfite-treated and PCR-amplified to distinguish CpG dinucleotides that are methylated from those that are not. Fluorescently labeled PCR products are hybridized to arrayed oligonucleotides that can discriminate between methylated and unmethylated alleles in regions of interest. Using this technique, two clinical subtypes of non-Hodgkin's lymphomas, mantle cell lymphoma, and grades I/II follicular lymphoma, were further separated based on the differential methylation profiles of several gene promoters. Work is underway in our laboratory to extend the interrogation power of this microarray system in multiple candidate genes. This novel tool, therefore, holds promise to monitor the outcome of various epigenetic therapies on cancer patients.     

Electroporation: basic principles, practical considerations and applications in molecular biology

There are many approaches available for introducing foreign DNA into eukaryotes and prokaryotes. The most commonly used technique and currently of more practical interest is the electroporation. This review will focus on the electroporation as a promising, highly efficient and effective means of gene transfer. We summarize a detailed assessment of the various parameters and conditions that govern electroporation of a wide range of cell types.     

DNA probes: applications of the principles of nucleic acid hybridization.

Nucleic acid hybridization with a labeled probe is the only practical way to detect a complementary target sequence in a complex nucleic acid mixture. The first section of this article covers quantitative aspects of nucleic acid hybridization thermodynamics and kinetics. The probes considered are oligonucleotides or polynucleotides, DNA or RNA, single- or double-stranded, and natural or modified, either in the nucleotide bases or in the backbone. The hybridization products are duplexes or triplexes formed with targets in solution or on solid supports. Additional topics include hybridization acceleration and reactions involving branch migration. The second section deals with synthesis or biosynthesis and detection of labeled probes, with a discussion of their sensitivity and specificity limits. Direct labeling is illustrated with radioactive probes. The discussion of indirect labels begins with biotinylated probes as prototypes. Reporter groups considered include radioactive, fluorescent, and chemiluminescent nucleotides, as well as enzymes with colorimetric, fluorescent, and luminescent substrates.     

An introduction to DNA chips: principles, technology, applications and analysis

This review describes the recently developed GeneChip technology that provides efficient access to genetic information using miniaturised, high-density arrays of DNA or oligonucleotide probes. Such microarrays are powerful tools to study the molecular basis of interactions on a scale that would be impossible using conventional analysis. The recent development of the microarray technology has greatly accelerated the investigation of gene regulation. Arrays are mostly used to identify which genes are turned on or off in a cell or tissue, and also to evaluate the extent of a gene's expression under various conditions. Indeed, this technology has been successfully applied to investigate simultaneous expression of many thousands of genes and to the detection of mutations or polymorphisms, as well as for their mapping and sequencing.     

Proteomics, nanotechnology and molecular diagnostics

Sequencing of the human genome opened the way to the exploration of the proteome and this has lead to the identification of large numbers of proteins in complex biological samples. The identification of diagnostic patterns in samples taken from patients to aid diagnosis is in the early stages of development. The solution to many of the technical challenges in proteomics and protein based molecular diagnostics will be found in new applications of nanomaterials. This review describes some of the physical and chemical principles underlying nanomaterials and devices and outlines how they can be used in proteomics; developments which are establishing nanoproteomics as a new field. Nanoproteomics will provide the platform for the discovery of next generation biomarkers. The field of molecular diagnostics will then come of age.     

Protein blotting: principles and applications

Extensive studies on the DNA tumor virus Simian Virus 40 (SV40) have provided a wealth of information regarding the genome organization, regulation of viral gene expression, and the mechanism of DNA replication. SV40 can grow lytically in permissive monkey cells or the viral DNA can integrate into the host genome of nonpermissive rodent cells causing morphological transformation. The viral DNA exists as a minichromosome within the nuclei of lytically infected cells and, as a consequence of DNA replication, there is a significant amplification of the viral genome during infection. These properties suggested that SV40 could be developed as a transducing vector to introduce exogenous DNA into mammalian cells and to express this foreign DNA during the SV40 infectious cycle. In this article the properties of SV40 virus vectors and SV40 hybrid plasmid vectors are described and contrasted.     

Nanoliposomes and their applications in food nanotechnology

Food nanotechnology involves the utilization of nanocarrier systems to stabilize the bioactive materials against a range of environmental and chemical changes as well as to improve their bioavailability. Nanoliposome technology presents exciting opportunities for food technologists in areas such as encapsulation and controlled release of food materials, as well as the enhanced bioavailability, stability, and shelf-life of sensitive ingredients. Liposomes and nanoliposomes have been used in the food industry to deliver flavors and nutrients and, more recently, have been investigated for their abilityto incorporate antimicrobials that could aid in the protection of food products against microbial contamination. In this paper, the main physicochemical properties of liposomes and nanoliposomes are described and some of the industrially applicable methods for their manufacture are reviewed. A summary of the application of nanoliposomes as carrier vehicles of nutrients, nutraceuticals, enzymes, food additives, and food antimicrobials is also presented.     

Single DNA molecule analysis: applications of molecular combing.

Dynamic changes to the genomic structure and to the DNA replication programme are important determinants of normal and abnormal cell development. To understand these changes and how they vary from cell to cell, single DNA molecules from both normal and abnormal cell populations must be examined and compared. Physical characterisation of single genomes at the kilobase level of resolution over large genomic regions is possible with molecular combing technology. An array of combed single DNA molecules is prepared by stretching molecules attached by their extremities to a silanised glass surface with a receding air-water meniscus. By performing fluorescent hybridisation on combed DNA, genomic probe position can be directly visualised, providing a means to construct physical maps and detect micro-rearrangements. Single-molecule DNA replication can also be monitored through fluorescent detection of incorporated nucleotide analogues on combed DNA molecules. These and other single-molecule applications of molecular combing are discussed in this paper and future developments of the technology are considered.     

Filamentous bacteriophage: biology, phage display and nanotechnology applications

Filamentous bacteriophage, long and thin filaments that are secreted from the host cells without killing them, have been an antithesis to the standard view of head-and-tail bacterial killing machines. Episomally replicating filamentous phage Ff of Escherichia coli provide the majority of information about the principles and mechanisms of filamentous phage infection, episomal replication and assembly. Chromosomally- integrated "temperate" filamentous phage have complex replication and integration, which are currently under active investigation. The latter are directly or indirectly implicated in diseases caused by bacterial pathogens Vibrio cholerae, Pseudomonas aeruginosa and Neisseria meningitidis. In the first half of the review, both the Ff and temperate phage are described and compared. A large section of the review is devoted to an overview of phage display technology and its applications in nanotechnology.     

Designed electromagnetic pulsed therapy: Clinical applications

The article to which this erratum refers was published in J Cell Phys (2007) 212: 579–582. © 2008 Wiley‐Liss, Inc.     

Designed electromagnetic pulsed therapy: clinical applications

First reduced to science by Maxwell in 1865, electromagnetic technology as therapy received little interest from basic scientists or clinicians until the 1980s. It now promises applications that include mitigation of inflammation (electrochemistry) and stimulation of classes of genes following onset of illness and injury (electrogenomics). The use of electromagnetism to stop inflammation and restore tissue seems a logical phenomenology, that is, stop the inflammation, then upregulate classes of restorative gene loci to initiate healing. Studies in the fields of MRI and NMR have aided the understanding of cell response to low energy EMF inputs via electromagnetically responsive elements. Understanding protein iterations, that is, how they process information to direct energy, we can maximize technology to aid restorative intervention, a promising step forward over current paradigms of therapy.     

Rational design of DNA sequences for nanotechnology, microarrays and molecular computers using Eulerian graphs

Nucleic acids are molecules of choice for both established and emerging nanoscale technologies. These technologies benefit from large functional densities of ‘DNA processing elements’ that can be readily manufactured. To achieve the desired functionality, polynucleotide sequences are currently designed by a process that involves tedious and laborious filtering of potential candidates against a series of requirements and parameters. Here, we present a complete novel methodology for the rapid rational design of large sets of DNA sequences. This method allows for the direct implementation of very complex and detailed requirements for the generated sequences, thus avoiding ‘brute force’ filtering. At the same time, these sequences have narrow distributions of melting temperatures. The molecular part of the design process can be done without computer assistance, using an efficient ‘human engineering’ approach by drawing a single blueprint graph that represents all generated sequences. Moreover, the method eliminates the necessity for extensive thermodynamic calculations. Melting temperature can be calculated only once (or not at all). In addition, the isostability of the sequences is independent of the selection of a particular set of thermodynamic parameters. Applications are presented for DNA sequence designs for microarrays, universal microarray zip sequences and electron transfer experiments.     

Towards applications of synthetic cells in nanotechnology

Synthetic cells, which are assembled anew from well-defined molecular parts, open-up new possibilities for nanotechnological applications due to their reduced complexity and high functionality. In this review, we discuss how synthetic cells are being implemented in different fields ranging from biomedicine to material science. On one hand, synthetic cells can serve as microreactors that house metabolic networks and as therapeutic carriers that directly communicate with living cells. On the other hand, synthetic cells can become active components in a new-generation of materials that process inputs and result in autonomous and adaptive behavior. These early examples highlight the potential impact that synthetic cells will have in future applications.     

Flow cytometry: basic principles and applications

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.