Screening for genetic mutations. A review

A point mutation of a nucleotide within a single gene can have a profound effect on a specific organ and/or the entire human body. DNA sequences associated with human diseases may differ from the corresponding normal sequences by single nucleotide mutations or by large alterations such as deletions, insertions, duplications, or translocations of DNA segments or entire chromosomes. As a result of the heterogeneity of DNA alterations and genetic mutations, various screening approaches are required to detect these alterations. However, methods which facilitate the detection of large mutations in the genome are typically insensitive to point mutations, whereas methods which detect point mutations are not appropriate to detect large alterations within the genome. Since there is no single perfect method to screen for unknown mutations, combinations of these methods may be necessary for accurate genetic diagnosis. The applications of polymerase chain reaction (PCR) technology to genomic screening have made rapid and accurate genetical diagnosis possible. Furthermore, recent developments in the technology of DNA microarrays have opened the way for high throughput sequence analysis by hybridization, which shows great potential in both molecular biology and medicine in the near future.     

The potential of electrophoretic mobility shift assays for clinical mutation detection

As the understanding of the links between genetic mutations and diseases continues to grow, there is an increasing need for techniques that can rapidly, inexpensively, and sensitively detect DNA sequence alterations. Typically, such analyses are performed on PCR-amplified gene regions. Automated DNA sequencing by capillary array electrophoresis can be used, but is expensive to apply to large numbers of patient samples and/or large genes, and may not always reveal low-abundance mutations in heterozygous samples. Many different types of genetic differences need to be detected, including single-base substitutions and larger sequence alterations such as insertions, deletions, and inversions. Electrophoretic mobility shift assays seem well suited to this purpose and could be used for the efficient screening of patient samples for sequence alterations, effectively reducing the number of samples that must be subjected to full and careful sequencing. While there is much promise, many of the mobility shift assays presently under development have yet to be demonstrated to have the high sensitivity and specificity of mutation detection required for routine clinical application. Hence, further studies and optimization are required, in particular the application of these methods not only to particular genes but also to large numbers of patient samples in blinded studies aimed at the rigorous determination of sensitivity and specificity. This review examines the state-of-the-art of the most commonly used mobility shift assays for mutation detection, including denaturing gradient gel electrophoresis, TGGE, SSCP, heteroduplex analysis, and denaturing HPLC.     

Simple electrochemical sensing of attomolar proteins using fabricated complexes with enhanced surface binding avidity

Various strategies have been proposed for the detection of disease protein biomarkers; however, most methods are too expensive, cumbersome or limited in sensitivity for clinical use. Here, we report that a fabricated complex can be used as a powerful tool to detect trace proteins in complex samples. In this strategy, a DNA–protein complex that comprises of one target molecule and two or more deoxyribozyme-containing probes can exhibit autonomous cleavage behavior on the surface of the substrate DNA modified electrode. In the meantime, the complex can remove the cleaved DNA fragment from the electrode surface by taking advantage of the proximity effect. The proposed approach allows one-step and highly sensitive detection of a variety of targets based on the changes of the direct electrochemical readout. Moreover, this method may also have considerable advantages over the commonly reported DNA amplification-assisted immunoassays, particularly in terms of assay simplicity and cost, which may hold great potential for application in resource-constrained regions.     

Terbium fluorescence as a sensitive,inexpensive probe for UV-induced damage in nucleic acids

Much effort has been focused on developing methods for detecting damaged nucleic acids. However, almost all of the proposed methods consist of multi-step procedures, are limited, require expensive instruments, or suffer from a high level of interferences. In this paper, we present a novel simple, inexpensive, mix-and-read assay that is generally applicable to nucleic acid damage and uses the enhanced luminescence due to energy transfer from nucleic acids to terbium(III) (Tb³⁺). Single-stranded oligonucleotides greatly enhance the Tb³⁺ emission, but duplex DNA does not. With the use of a DNA hairpin probe complementary to the oligonucleotide of interest, the Tb³⁺/hairpin probe is applied to detect ultraviolet (UV)-induced DNA damage. The hairpin probe hybridizes only with the undamaged DNA. However, the damaged DNA remains single-stranded and enhances the intrinsic fluorescence of Tb³⁺, producing a detectable signal directly proportional to the amount of DNA damage. This allows the Tb³⁺/hairpin probe to be used for sensitive quantification of UV-induced DNA damage. The Tb³⁺/hairpin probe showed superior selectivity to DNA damage compared to conventional molecular beacons probes (MBs) and its sensitivity is more than 2.5 times higher than MBs with a limit of detection of 4.36 ± 1.2 nM. In addition, this probe is easier to synthesize and more than eight times cheaper than MBs, which makes its use recommended for high-throughput, quantitative analysis of DNA damage.     

The review of Lab-on-PCB for biomedical application

Prevention of infectious diseases, diagnosis of diseases, and determination of treatment options all rely on biosensors to detect and analyze biomarkers, which are usually divided into four parts: cell analysis, biochemical analysis, immunoassay, and molecular diagnosis. However, traditional biosensing devices are expensive, bulky, and require a lot of time to detect, which also limited its application in resource-limited areas. In recent years, Lab-on-PCB, which combines biosensing technology and PCB technology, has been widely used in biomedical applications due to its high integration, personalized design, and easy mass production. Among these Lab-on-PCB sensing devices, the PCB circuit plays an important role. It can be directly used as a resistance sensor to count cells, and also used as a control device to automatically control the detection device. Flexible PCBs can be used to make wearable medical biosensors. In addition, due to the high degree of integration of the PCB circuit, Lab-on-PCB can perform multiple inspections on the same platform, which reduces the inspection time equivalently. Therefore, in this review paper, we discuss the application of Lab-on-PCB in four analysis methods of cell analysis, biochemical analysis, immunoassay, and molecular diagnosis, and give some suggestions for improvement and future development trends at the end.     

Molecular beacon probes for the detection of cisplatin-induced DNA damage

Cisplatin (cis-diamminedichloroplatinum(II)) causes crosslinking of DNA at AG and GG sites in cellular DNA, inhibiting replication, and making it a useful anti-cancer drug. Several techniques have been used previously to detect nucleic acid damage but most of these tools are labour-intensive, time-consuming, and/or expensive. Here, we describe a sensitive, robust, and quantitative tool for detecting cisplatin-induced DNA damage by using fluorescent molecular beacon probes (MB). Our results show a decrease of fluorescence in the presence of cisplatin-induced DNA damage, confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The decrease in fluorescence upon damage scales with the number of AG and GG sites, indicating the ability of MB to quantitatively detect DNA damage by cisplatin.     

DNA typing in hereditary disease.

An increasing number of hereditary diseases are becoming amenable to diagnosis by analysis of DNA as the responsible genes are located and identified. Gel electrophoresis of DNA fragments plays a central role in the diagnosis of hereditary disease. Electrophoretic separation of differently sized fragments enables the characterization or typing of normal variants which are known to be genetically linked to disease genes. For some diseases it is possible to directly detect mutations by DNA electrophoresis. Deletion mutants may be detected by a restriction fragment of altered size or by a failure to amplify a coding region with the polymerase chain reaction. Carriers of small deletions, involving a few base pairs, may be identified by DNA amplification which produces heteroduplexes that show characteristic, anomalous electrophoretic migration. Mutations that alter restriction sites also alter the sizes of restriction fragments. Common disease mutations that alter a single base pair may be detected using a pair of reactions with normal and mutant oligonucleotides under conditions where a perfect match is necessary for hybridization, amplification or ligation. Alternatively a mismatched oligonucleotide primer may be designed to generate a restriction site with either the normal or mutant allele, following DNA amplification. Finally a number of techniques are available that are useful as screening tools for novel mutations.     

Versatile microfluidic complement fixation test for disease biomarker detection

The complement fixation test (CFT) is a serological test that can be used to detect the presence of specific antibodies or antigens to diagnose infections, particularly diseases caused by microbes that are not easily detected by standard culture methods. We report here, for the first time, a poly(dimethylsiloxane) (PDMS)/glass slide hybrid microfluidic device that was used to manipulate the solution compartment and communication within the microchannel to establish sampler and indicator systems of CFT. Two types of on-chip CFT, solution-based and solid phase agar-based assays, were successfully demonstrated for biomarker carcinoembryonic antigen (CEA) and recombinant avian influenza A (rH7N9) virus protein detection. In addition, the feasibility of the on-chip CFT in assaying real biopsy was successfully demonstrated by specifically detecting rH7N9 and CEA in human serum. The results demonstrated that the miniaturized assay format significantly reduced the assay time and sample consumption. Exemption from protein immobilization, blocking, complicated washing steps and expensive enzyme/fluorescein conjugates highlights the merits of on-chip CFT over ELISA. Most attractively, the on-chip agar-based CFT results can be imaged and analysed by smartphone, strengthening its point-of-care application potential. We anticipate that the on-chip CFT reported herein will be a useful supplemental or back-up tool for on-chip immunoassays such as ELISA for disease diagnosis and food inspection.     

PCR-based detection of gene transfer vectors: application to gene doping surveillance

Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.     

Electrochemical Determination of Natural Drug Colchicine in Pharmaceuticals and Human Serum Sample and its Interaction with DNA

Colchicine (COLC) is a natural toxic product and secondary metabolite most commonly used to treat gout. In this study, its electrochemical behavior and determination was investigated by employing modification‐free boron‐doped diamond electrode (BDDE). Besides, its interaction with DNA was monitored using electrochemical methods. It was found that oxidation of this compound proceeds in two steps, where first sharp and well defined oxidation peak occurs at potential of around 1.19 V, and second one at around 1.37 V, in Britton‐Robinson buffer solution at pH 7.5. Wide dynamic range from 1 to 100 μM was obtained with a detection limit (3σ_intercept/slope) of a 0.26 μM, based on the evaluation of first oxidation peak using differential pulse voltammetry. The proposed method was also found to be suitable for monitoring interaction of this drug with DNA as important segment for medical use. Concerning the validation, the analytical procedure shows excellent selectivity and sensitivity toward COLC detection and after method development it was successfully used for its quantification in pharmaceutical preparation and human serum sample, with satisfactory recovery. Obviously, this approach can be promising replacement for time‐consuming and expensive separation methods.     

Quantification of DNA in forensic samples

Quantification of DNA in a forensic sample is of major importance for proper DNA amplification and STR profiling. Several methods have been developed to quantify DNA, from basic UV spectrometry, through gel-based techniques, to dye staining, blotting techniques, and, very recently, DNA amplification methods (polymerase chain reaction, PCR). Early techniques simply measured total DNA, but newer techniques can specifically measure human DNA while excluding non-human DNA (foodstuff, animal, or bacterial contamination). These newer assays can be faster and less expensive than traditional methods, making them ideal for the busy forensic laboratory. This paper reviews classic and newer quantification techniques and presents methods recently developed by the authors on the basis of PCR of Alu sequences.     

Review of denaturant capillary electrophoresis in DNA variation analysis

Analyses of germline and somatic single-nucleotide DNA variations are important in both population genetics research and clinical practice. Reliable and inexpensive methods that are flexible and designed for automation are required for these analyses. Present day DNA sequencing technology is too expensive for testing all 22-25 000 human genes in populations genetics studies or in scanning large numbers of tumors for novel mutations. Denaturant capillary electrophoresis (DCE) has the potential to meet the need for large-scale analysis of DNA variants. Several different analyses can be performed by DCE, including mutation analysis, single-nucleotide polymorphism (SNP) discovery in individual and pooled samples, detection of allelic imbalance, and determination of microhaplotypes. Here we review the theoretical background of the method, its sensitivity, specificity, detection limit, throughput, and repeatability in the light of current literature in the field.     

Determination of Mercury with a Miniature Sensor for Point-of-care Testing

In developing countries, subsistence gold mining entails mixing metallic mercury with crushed sediments to extract gold. In this approach, the gold−mercury amalgam is heated to evaporate mercury and obtain gold. Thus, the highly volatile mercury can be absorbed through inhalation, resulting in adverse health effects. Urinalysis can be used to detect mercury, which is excreted in urine and feces, and correlate exposure with toxic effects. The current gold standard analytical methods are based on fluorescence or inductively coupled plasma mass spectrometry methods, but are expensive, time consuming, and are not easily accessible in countries where testing is needed. In this work, we report on a miniature electrochemical sensor that can rapidly detect mercury in urine at levels well below the US Biological Exposure Index (BEI) limit of 50 ppb (μg/L). The sensor is based on a thin-film gold electrode and anodic stripping voltammetry electroanalytical approach. The sensor successfully detected mercury at trace levels in urine, with a limit of detection of ∼15 ppb Hg in the linear range of 20–80 ppb. With the low-cost disposable sensors and portable instrumentation, it is well suited for point-of-care applications.     

The effect of heat treatment on the detection of peanut allergens as determined by ELISA and real-time PCR

Peanut allergic reactions can result from the ingestion of even very small quantities of peanut and represent a severe threat to the health of sensitised individuals. The detection of peanut traces in food products is therefore of prime importance. Peanut traces which can be (unintentionally) present in food products have usually undergone one or more processing steps like roasting and baking. Therefore, methods designed to detect such traces have to be capable of detecting heat-treated peanuts. Commonly used methodologies designed to detect peanut traces in food products are enzyme-linked immunosorbent assays (ELISAs) that detect peanut-specific proteins, and polymerase-chain-reaction (PCR)-based methods targeting peanut-specific DNA. A comparative analysis of such methods was performed and the impact of heat treatment on peanut kernels as well as the impact on a peanut-containing food matrix are investigated. Our results show that heat treatments have a detrimental effect on the detection of peanut with either type of method and that both types of methods are affected in a similar manner.     

A Double‐Imprinted Diffraction‐Grating Sensor Based on a Virus‐Responsive Super‐Aptamer Hydrogel Derived from an Impure Extract

The detection of viruses is of interest for a number of fields including biomedicine, environmental science, and biosecurity. Of particular interest are methods that do not require expensive equipment or trained personnel, especially if the results can be read by the naked eye. A new “double imprinting” method was developed whereby a virus‐bioimprinted hydrogel is further micromolded into a diffraction grating sensor by using imprint‐lithography techniques to give a “Molecularly Imprinted Polymer Gel Laser Diffraction Sensor” (MIP‐GLaDiS). A simple laser transmission apparatus was used to measure diffraction, and the system can read by the naked eye to detect the Apple Stem Pitting Virus (ASPV) at concentrations as low as 10 ng mL⁻¹, thus setting the limit of detection of these hydrogels as low as other antigen‐binding methods such as ELISA or fluorescence‐tag systems.     

Oxidation Chemistry of DNA and p53 Tumor Suppressor Gene

The chemistry of DNA and its repair selectivity control the influence of genomic oxidative stress on the development of serious disorders such as cancer and heart diseases. DNA is oxidized by endogenous reactive oxygen species (ROS) in vivo or in vitro as a result of high energy radiation, non-radiative metabolic processes, and other consequences of oxidative stress. Some oxidations of DNA and tumor suppressor gene p53 are thought to be mutagenic when not repaired. For example, site-specific oxidations of p53 tumor suppressor gene may lead to cancer-related mutations at the oxidation site codon. This review summarizes the research on the primary products of the most easily oxidized nucleobase guanine (G) when different oxidation methods are used. Guanine is by far the most oxidized DNA base. The primary initial oxidation product of guanine for most, but not all, pathways is 8-oxoguanine (8-oxoG). With an oxidation potential much lower than G, 8-oxoG is readily susceptible to further oxidation, and the products often depend on the oxidants. Specific products may control the types of subsequent mutations, but mediated by gene repair success. Site-specific oxidations of p53 tumor suppressor gene have been reported at known mutation hot spots, and the codon sites also depend on the type of oxidants. Modern methodologies using LC–MS/MS for codon specific detection and identification of oxidation sites are summarized. Future work aimed at understanding DNA oxidation in nucleosomes and interactions between DNA damage and repair is needed to provide a better picture of how cancer-related mutations arise.     

Colorimetric detection of hepatitis B virus (HBV) DNA based on DNA-templated copper nanoclusters

DNA detection plays an important role in early diagnosis of genetic disease. The conventional detection methods of DNA are based on expensive equipment, which do not meet the demands of developing countries. Thus, we developed a colorimetric method, which could be observed with naked eye and used copper nanoclusters for cost-effective. Moreover, the target of this method is the DNA in Hepatitis B virus that is one of the most popular chronic viral infections in developing countries over the past years. Our method was sensitive and the limit of detection was 12 × 10⁹ molecules. Three-base-pair mismatches target DNA was detected easily. These results revealed the favorable sensitivity and selectivity of this approach. Most importantly, our method may have potential applications in correct diagnosis of genetic disease and monitoring of gene therapy in the poverty-stricken areas.     

Application of medical and analytical methods in Lyme borreliosis monitoring

Lyme borreliosis (LB) is one of the most common tick-borne diseases in the northern hemisphere. It is a chronic inflammatory disease caused by the spirochaete Borrelia burgdorferi. In its early stages, pathological skin lesions, namely erythema chronicum migrans, appear. The lesions, usually localised at the site of the bite, may become visible from a few weeks up to 3 months after the infection. Predominant clinical symptoms of the disease also involve joint malfunctions and neurological or cardiac disorders. Lyme disease, in all its stages, may be successfully treated with antibiotics. The best results, however, are obtained in its early stages. In order to diagnose the disease, numerous medical or laboratory techniques have been developed. They are applied to confirm the presence of intact spirochaetes or spirochaete components such as DNA or proteins in tick vectors, reservoir hosts or patients. The methods used for the determination of LB biomarkers have also been reviewed. These biomarkers are formed during the lipid peroxidation process. The formation of peroxidation products generated by human organisms is directly associated with oxidative stress. Apart from aldehydes (malondialdehyde and 4-hydroxy-2-nonenal), many other unsaturated components such as isoprostenes and neuroprostane are obtained. The fast determination of these compounds in encephalic fluid, urine or plasma, especially in early stages of the disease, enables its treatment. Various analytical techniques which allow the determination of the aforementioned biomarkers have been reported. These include spectrophotometry as well as liquid and gas chromatography. The analytical procedure also requires the application of a derivatization step by the use of selected reagents.