DNA damage in a solution containing copper(II) ions and ascorbic acid: Effect of the presence of sulfite

Some antioxidant compounds have a pro-oxidant effect in the presence of transition metal ions, due to the reduction of Mn+ to M(n-1)+ with simultaneous formation of free radicals, which then promote DNA damage. In the present study, we evaluated the pUC19 DNA damage in a solution containing Cu(II) and ascorbic acid (AA) or S(IV) saturated with air by agarose gel electrophoresis. Our results showed that this damage decreases if AA and S(IV) are simultaneously added. This study also illustrates the importance of Cu(II) in this process, as no DNA damage was observed when AA or S(IV) were present in the absence of this metallic ion. Our data showed that DNA preservation depends on the concentration of AA and S(IV) and occurs when the [S(IV)]:[AA] ratio ranges from 1:1 to 20:1. Absorbance measurements and thermodynamic data show that no reaction occurs between AA and S(IV) when this mixture (pH 5.5) is added to pUC-19 DNA. The presence of dissolved oxygen may be the cause of AA consumption in the mixture of these two antioxidants, which subsequently decreases DNA damage.

A low-cost smart system for electrophoresis-based nucleic acids detection at the visible spectrum.

Nucleic acid detection by electrophoresis is still a quick and accessible technique for many diagnosis methods, primarily at research laboratories or at the point of care units. Standard protocols detect DNA/RNA molecules through specific bound chemical dyes using a UV-transilluminator or UV-photo documentation system. However, the acquisition costs and availability of these devices, mainly the ones with photography and internet connection capabilities, can be prohibitive, especially in developing countries public health units. Also, ultraviolet radiation is a common additional risk factor to professionals that use electrophoresis-based nucleic acid detection. With that in mind, this work describes the development of a low-cost DNA/RNA detection smart system capable of obtaining qualitative and semi-quantitative data from gel analysis. The proposed device explores the visible light absorption range of commonly used DNA/RNA dyes using readily available parts, and simple manufacturing processes, such as light-emitting diodes (LEDs) and 3D impression. By applying IoT techniques, our system covers a wide range of color spectrum in order to detect bands from various commercially used dyes, using Bluetooth communication and a smartphone for hardware control, image capturing, and sharing. The project also enables process scalability and has low manufacturing and maintenance costs. The use of LEDs at the visible spectrum can achieve very reproducible images, providing a high potential for rapid and point-of-care diagnostics as well as applications in several fields such as healthcare, agriculture, and aquaculture.

Immunoelectrophoresis: A Method with Many Faces.

Immunoelectrophoresis (IEP) was the first practical method that combined electrophoresis and immunoprecipitation for identifying and characterizing proteins within complex mixtures. Over the years, IEP has been extended to include a variety of techniques and, as a general name, has been applied to virtually any technique that involves electrophoresis and antigen-antibody precipitin reaction for proteins. Because of the diversity in technical details of different IEP versions, the method described here deals only with classic IEP. Although it requires some manual expertise, IEP is versatile, relatively easy to customize, and economical with no need for expensive instrumentation. Further, it can discern identity, partial identity, and nonidentity of the proteins. Any low-viscosity body fluid specimen or, possibly, culture fluid and tissue extract could be tested with IEP if proper antibodies are available. With these attributes, classic IEP remains a valuable tool for clinical diagnostic testing, purity checking of biochemical and pharmaceutical products, and research.

Professor Volodymyr Oleksiiovych Shlyakhovenko (on the 80th birth anniversary).

In September 2018, Professor Volodymyr Oleksiiovych Shlyakhovenko, well-known Ukrainian scientist in the field of cancer biochemistry, celebrated his 80th anniversary.

Gene expression studies using a miniaturized thermal cycler system on board the International Space Station.

The distance and duration of human spaceflight missions is set to markedly increase over the coming decade as we prepare to send astronauts to Mars. However, the health impact of long-term exposure to cosmic radiation and microgravity is not fully understood. In order to identify the molecular mechanisms underpinning the effects of space travel on human health, we must develop the capacity to monitor changes in gene expression and DNA integrity in space. Here, we report successful implementation of three molecular biology procedures on board the International Space Station (ISS) using a miniaturized thermal cycler system and C. elegans as a model organism: first, DNA extraction-the initial step for any type of DNA analysis; second, reverse transcription of RNA to generate complementary DNA (cDNA); and third, the subsequent semi-quantitative PCR amplification of cDNA to analyze gene expression changes in space. These molecular procedures represent a significant expansion of the budding molecular biology capabilities of the ISS and will permit more complex analyses of space-induced genetic changes during spaceflight missions aboard the ISS and beyond.

Kinetic Analysis of the Exonuclease Activity of the Bacteriophage T4 Mre11-Rad50 Complex.

Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that is responsible for host genome degradation and the processing of DNA ends for recombination-dependent DNA repair. In this chapter, we describe the ensemble methods currently employed by our laboratory to characterize the exonuclease activity of the T4 MR complex. DNA exonucleases play a vital role in maintaining the integrity of DNA through their participation in DNA repair pathways and as proofreaders for DNA polymerases. Methods for quantifying the general features of the exonuclease, and for determining steady-state kinetic parameters (Km, kcat), the polarity of exonuclease activity, and processivity are presented. These methods should be applicable to all DNA exonucleases, and to some extent endonucleases.

Development of bufferless gel electrophoresis chip for easy preparation and rapid DNA separation.

This work presents a handy, fast, and compact bufferless gel electrophoresis chip (BGEC), which consists of precast agarose gel confined in a disposable plastic body with electrodes. It does not require large volumes of buffer to fill reservoirs, or the process of immersing the gel in the buffer. It withstands voltages up to 28.4 V/cm, thereby allowing DNA separation within 10 min with a similar separation capability to the standard gel electrophoresis. The results suggest that our BGEC is highly suitable for in situ gel electrophoresis in forensic, epidemiological settings and crime scenes where standard gel electrophoresis equipment cannot be brought in while quick results are needed.

Twelve-Gel Comet Assay Format for Quick Examination of DNA Damage and Repair.

The comet assay (single cell gel electrophoresis) is a sensitive, versatile method for detecting DNA damage in eukaryotic cells. The traditional comet assay format has 1 or 2 gels on a microscope slide, 1 sample per slide, and there is a limit of 40 gels per experiment given the size of a typical electrophoresis tank. To increase throughput, we have designed and tested a system with 12 minigels on one slide, allowing analysis of up to 12 times more samples in one electrophoresis run. The novel comet assay format compares well with the traditional technology. The various steps are suitable for further automation, and the formats can be adapted to fully automated scoring. The new procedures save time at all stages as fewer slides are handled, and the amounts of reagents needed are reduced significantly. This format is particularly useful for testing of numerous genotoxic agents and nanomaterials at different concentrations and on different types of cells; simultaneous analysis of different lesions using a range of enzymes; and analysis of cell extracts for DNA repair activity.

Multilocus Sequence Typing (MLST) for Cronobacter spp.

MLST is a molecular typing technique that involves the identification and clustering of bacterial isolates based on the partial sequence analysis of multiple housekeeping genes (generally seven) which are distributed across the length of the genome of the organism. The Cronobacter whole genus MLST scheme can be successfully used for an accurate species level identification and classification of this complex genus.

Restriction Endonuclease Analysis Typing of Clostridium difficile Isolates.

Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium difficile (CD) isolates. REA typing analyzes CD whole cellular DNA on two levels of discrimination: REA Group designation and REA Type designation, which distinguishes specific subtypes within the REA Group. This methodology has enabled the tracking of epidemiologically significant CD strains over time and in some cases has allowed documentation of the evolution of previously rare REA Group strains that have subsequently become epidemic. The chapter details the methods used to isolate and purify CD colonies from stool samples, to obtain intact, full-length whole cellular DNA from CD isolates by use of guanidine-EDTA solution, and to analyze the HindIII-digested DNA after electrophoretic separation on agarose gels.

Direct PCR-Ribotyping of Clostridium difficile.

PCR-ribotyping, a method based on heterogeneity of ribosomal intergenic spacer region, is the preferred method for genotyping of Clostridium difficile. Standardly used procedure for PCR-ribotyping is culturing of C. difficile from fecal samples and subsequent typing. In this chapter, we describe a modified PCR-ribotyping method for direct detection of PCR-ribotypes directly in total stool DNA extract, without prior need to isolate C. difficile.

Why Johnny can't clone: Common pitfalls and not so common solutions.

The demand for cloned genes has increased incessantly over the past 32 years, but some who need recombinant plasmids struggle to produce them. While the pitfalls of traditional ligation-dependent cloning are non-trivial, most can be avoided with sufficient effort and attention to detail. Here, the chemical properties of enzymes and reagents used to clone genes into plasmids are reviewed to draw attention to the most pertinent details. In particular, the virtues of agarose gel electrophoresis monitoring, the nature of the interactions between DNA and silica, and challenges associated with thermostable DNA polymerases, restriction endonucleases, and T4 DNA ligase are explored. Common pitfalls associated with Escherichia coli transformation and DNA modifying enzymes are also described. A thorough understanding of established methods is essential for troubleshooting, implementing alternative approaches, and inventing new techniques in response to changes in technology and demand.

Blood grouping based on PCR methods and agarose gel electrophoresis.

The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

Low electric field DNA separation and in-channel amperometric detection by microchip capillary electrophoresis.

Miniaturisation of microchip capillary electrophoresis (MCE) is becoming an increasingly important research topic, particularly in areas related to micro total analysis systems or lab on a chip. One of the important features associated with the miniaturised MCE system is the portable power supply unit. In this work, a very low electric field MCE utilising an amperometric detection scheme was designed for use in DNA separation. The device was fabricated from a glass/polydimethylsiloxane hybrid engraved microchannel with platinum electrodes sputtered onto a glass substrate. Measurement was based on a three-electrode arrangement, and separation was achieved using a very low electric field of 12 V/cm and sample volume of 1.5 µl. The device was tested using two commercial DNA markers of different base pair sizes. The results are in agreement with conventional electrophoresis, but with improved resolution. The sensitivity consistently higher than 100 nA, and the separation time approximately 45 min, making this microchip an ideal tool for DNA analysis.

Northern blotting.

To measure the size and amount of RNA transcribed from a specific gene of interest.

The use of the MegaBACE for sequencing and genotype analysis.

Despite the advent of next generation sequencing techniques, which provide access to an enormous amount of genomic information in a relatively short time, the conventional Sanger sequencing and microsatellite genotyping analyses present a straightforward method to answer clearly defined questions in population genetics, phylogeography, or forensics. The MegaBACE is a platform that provides both applications with equally reliable performance. In this overview, protocols for the classical techniques of Sanger sequencing and microsatellite genotyping are described. This chapter aims to supply the user of the MegaBACE with methodological tools and some "insider" knowledge of this highly sensitive apparatus.

von Willebrand Factor multimers profiling with a semi-automated system.

Abnormalities in plasma von Willebrand factor (vWF) concentration and function result in von Willebrand disease (vWD). The diagnosis requires a battery of tests such as screening procedures, confirmatory tests, phenotypic characterization, and genotyping. The phenotypic testing (multimer pattern analysis) is important in order to subclassify the hereditary and the acquired forms of vWD. Only few laboratories are skilled to perform this analysis. The extreme range of protein size from 250 kDa monomer to over 20,000 kDa multimers requires a time-consuming procedure (3-4 days) and presents many technical difficulties. To standardize the method and to overcome technical difficulties, we developed a rapid and sensitive semi-automated method to visualize the multimeric structure of vWF. The semi-automated method we present performs the electrophoresis of patient's plasma in 120 min on a precast gel. Gels are suitable for the G26 Interlab instrumentation. After gel blotting, the method allows visualization of the vWF multimer pattern directly on the membrane. We reduced the time required from 72 to 8 h and we propose this test for the first level screening of vWF multimer deficiency.

A simple monolithic column electroelution for protein recovery from gel electrophoresis.

Protein recovery from gel electrophoresis plays an important role in functional genomics and proteomics but faces a series of issues (e.g., complex procedure, low recovery, long experimental time). In this study, a monolithic column electroelution (MCE) was developed for protein recovery from gel electrophoresis. With the model proteins of bovine serum albumin (BSA), hemoglobin (Hb), and myoglobin (Mb), the developed device and method were compared with common electroelution procedures in agarose gel electrophoresis (AGE). The comparative experiments revealed that (i) the protein recovery achieved with the developed device was greater than 83%, much higher than the 41% to 50% achieved with the common devices; (ii) the running time to obtain 70% recovery was approximately 15 min, evidently shorter than the 240 min with the common devices; and (iii) the device and procedure were simple and less time-consuming as compared with those of the common devices. It was observed that the serum protein bands cut from polyacrylamide gel electrophoresis could be transferred into solution in 15 to 30 min with 82% yield. The device, along with its relevant procedure, has potential use in protein extraction and proteomics as well as in DNA studies.

[The comparative analysis of electrophoretic fractionating of blood serum proteins in diagnostics of multiple plasma cell myeloma].

The choice of technology of electrophoretic fractionating of blood serum proteins is determined, besides the analytical characteristics, by its economic component. The electrophoresis technologies developed by the R&D production facility "Astra" (Russia) and the firm "PZ Cormay S.A." (Poland) are compared from a viewpoint of applicability in routine laboratory, practice and diagnostics of multiple plasma cell myeloma in particular. It is established that under the comparable economic, "consumer" and analytic characteristics of technologies in the diagnostic process the application of the technology in agarose gel ("PZ Cormay S.A.") is more preferable.