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Development of a Peptide Nucleic Acid Array Platform for the Detection of Genetically Modified Organisms in Food
Abstract
Two previously developed platforms, a multiplex polymerase chain reaction (PCR) and a peptide nucleic acid (PNA) array, the former allowing for the simultaneous detection of five transgenes and two endogenous controls in food and feed matrices and the latter for the assessment of the identity of amplified PCR products, were combined in order to develop a PNA array device for the screening of genetically modified organisms (GMOs) in food. PNA probes were opportunely designed, synthesized, and deposited on commercial slides. The length of the probes as well as the distance of the probes from the surface were evaluated and found to be critical points. The most suitable probes were found to be 15-mer PNAs linked to the slide surface by means of two 2-(2-aminoethoxy)ethoxyacetic acids as spacers. The device was tested on a model system constituted by flour samples containing a mixture of standards at known concentrations of transgenic material, in particular Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize: The DNA was amplified using the specific multiplex PCR method and tested on the PNA array. The method proposed was found to be able to correctly identify every GMO present in the tested samples.
... Generally peptide backbone is formed from N-aminoethylglycine monomers [158] and these molecules can hybridize very strongly to complementary DNA and RNA sequences [159]. Peptide nucleic acid (PNA) arrays have also been developed to be used, oft en in combination with muliplex PCRs, for GMO screening [160,161]. Germini et al. [161] have designed PNA probes to screen seven DNA target sequences amplifi ed by m-PCR (MON810, Bt11, Bt176, GA21 maize events, RR soybean events, zein and lectin genes). Hamels et al. [162] have screened 24 transgenic events and identifi ed them with 0.1% limit of detection using m-PCR-microarray method. ...
... Peptide nucleic acid (PNA) arrays have also been developed to be used, oft en in combination with muliplex PCRs, for GMO screening [160,161]. Germini et al. [161] have designed PNA probes to screen seven DNA target sequences amplifi ed by m-PCR (MON810, Bt11, Bt176, GA21 maize events, RR soybean events, zein and lectin genes). Hamels et al. [162] have screened 24 transgenic events and identifi ed them with 0.1% limit of detection using m-PCR-microarray method. ...
... 19,20 On account of these properties, PNAs have been used, in combination with different analytical techniques, in a wide variety of diagnostic methods for the detection of specific DNA tracts in biomedical and food research. 21 In particular, PNA microarray technology proved to be very effective for the detection of genetically modified organisms (GMOs) 22,23 or hidden allergens in food. 24 A PNA microarray platform was also recently developed for the detection of SNPs related to tomato cultivars. ...
... However, due to the very low signal intensity, optimization of the entire process from oil to PNA array analysis should be performed. Ultrasensitive PNA-based techniques, allowing to selectively detect SNPs and point mutations with only few copies of DNA, 22,23 can greatly improve this type of analysis. ...
PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.
... The presence of self-paired sequences was also minimized since these are known to prevent interaction with complementary DNA. Two 2-(2-aminoethoxy)ethoxyacetic acid (AEEA) linkers were added at the N terminus of PNA probes to increase the accessibility of the target DNA, according to our previous studies using microarray technology (Germini et al., 2005). All PNAs were produced using automatic solid-phase synthesis with standardized protocols (Manicardi et al., 2018), purified by HPLC and characterized by UPLC-MS, as described previously (Mejia-Ariza et al., 2017), for purity and identity assessment (see Supplementary Figs. ...
RAS mutations in the blood of colorectal cancer (CRC) patients are emerging as biomarkers of acquired resistance to Epidermal Growth Factor Receptor therapy. Unfortunately, reliable assays granting fast, real-time monitoring of treatment response, capable of refining retrospective, tissue-based analysis, are still needed. Recently, several methods for detecting blood RAS mutations have been proposed, generally relying on multi-step and PCR-based, time-consuming and cost-ineffective procedures. By exploiting a liquid biopsy approach, we developed an ul-trasensitive nanoparticle-enhanced plasmonic method for detecting ~1 aM RAS single nucleotide variants (SNVs) in the plasma of CRC patients. The assay does not require the extraction of tumor DNA from plasma and detects it in volumes as low as 40 μL of plasma, which is at least an order of magnitude smaller than that required by state of the art liquid biopsy technologies. The most prevalent RAS mutations are detected in DNA from tumor tissue with 100% sensitivity and 83.33% specificity. Spike-in experiments in human plasma further encouraged assay application on clinical specimens. The assay was proven in plasma from CRC patients and healthy donors, and full discrimination between mutated DNA from patients over wild-type DNA from healthy volunteers was obtained thus demonstrating its promising avenue for cancer monitoring based on liquid biopsy.
... This technology is based on the visualization of hybridization reactions between nucleic acids in a sample with unique nucleic acid sequences of known pathogens immobilized on a solid surface. Microarrays can detect up to thousands of unique sequences, and the range of organisms detected can be expanded to include not only all the different pathogens which can affect a host plant but also specific plant features, such as the variety or the presence of GM traits (Germini et al. 2005). However, production of hybridisation signals can be highly variable and extremely sensitive to minor technical differences (Carmichael 2012). ...
The main goal of seed pathology research and practice is the production and dissemination of high-quality, disease-free seed that maximizes potential crop productivity and value. Presently, the largest part of official seed health tests requires the growth of pathogens (direct methods) but molecular biology offers new tools to diagnose fungal pathogens in/on seeds (indirect methods), reducing the time required by direct methods and improving the output of seed health tests. However, molecular methods suffer some drawbacks and require a more difficult validation procedure than direct methods. This chapter aims at describing the historical context, and the development and implementation of methods for the detection and monitoring of seed-borne plant pathogenic fungi in Europe, with special emphasis on innovative methods. © 2014 Springer Science+Business Media Dordrecht. All rights are reserved.
... Main advantages of microarrays are miniaturization, high sensitivity and screening throughput. Different DNA approaches coupled with multiplex PCR have been reported: multiplex DNA array-based PCR for quantification of GM maize; ligation detection reaction coupled with universal array technology for detection of GM maize event Bt11; peptide nucleic acid array approach for detection of five GM events and two plant species; multiplex DNA microarray chip for simultaneous identification of nine GMOs, five plant species and three screening elements, namely, p35S, Tnos and nptII; an event-specific DNA microarray system to detect 19 GM events, two of soybean, thirteen of maize, three of canola and one of cotton, in processed foods [62][63][64][65][66] . ...
Efficient detection strategies for genetically modified (GM) crops need to be in compliance with regulatory frameworks and address consumer concerns. The present review describes widely employed DNA-based technologies for GM detection. Polymerase chain reaction (PCR) and real-time PCR (qPCR) are the methods that can be used for qualitative and quantitative analysis of GM crops due to their specificity, sensitivity and robustness. With increase in number and complexity of genetic elements in newly developed GM events, strategies based on matrix approach, real-time PCR-based multi-target system, loop-mediated iso-thermal amplification, next generation sequencing, have emerged, which could facilitate cost-effective, rapid, on-site or high throughput GM detection.
... As these problems become resolved it may be possible in the future to combine the detection of broader ranges of different types of pathogens from large host ranges on single arrays. It may also be possible to include the screening of other traits in plants, such as variety, presence of GM events (Germini et al., 2005;Rudi et al., 2003) or even the expression of genes caused by environmental stress that could be confused with pathogen infection (Tamaoki et al., 2004). ...
... The method is demonstrated that PNA array is efficient for simultaneous detections. Its sensitivity is however affected from the length of probes and their distance from the slide surface (Germini et al., 2005). ...
... The method is demonstrated that PNA array is efficient for simultaneous detections. Its sensitivity is however affected from the length of probes and their distance from the slide surface (Germini et al., 2005). ...
... Also, microarray assay and nanotechnology as new approaches for detection methods for GM plants were investigated.Onishi et al. 2005; Shrestha et al. 2008; Kim et al. 2006b)과 최근에 추가 적인 4개 이벤트인 Event3272, LY038, MIR162, and MON88017 (Kim et al. 2009b)와 GM 콩의 4개 이벤트인 RRS, A2704-12, DP356043-5, MON89788 (Kim et al. 2009c), GM 카놀라의 경우 3개의 이벤트 GT73, MS8xRF3, T45 (Kim et al. 2007), GM 면화의 경우 4개의 이벤트인 MON1445, MON15985, MON88913, LLcotton25 (Kim et al. 2008Table 2)하여 표준단백질로 활용하여 단백질의 정량검사에 활 용할 수 있다. 이와 같이 GMO 분석의 새로운 기술로써 microarray을 이용한 사례로 GM 옥수수와 콩의 검사를 위한 DNA microarray-multiplex PCR법이 보고된 바 있으며 (Germini et al. 2005; Bordoni et al. 2005), 9개의 GM event (Bt176, Bt11, GA21, MON810, CBH351, T25, Topas 19/2, T45, RRS) 의 검출을 위한 low-density DNA chip이 보고된 바 있다 (Leimanis et al. 2006). 우리나라에서는 가공식품 내에서 GMO 검정을 하기 위해 2개의 GM 콩, 13개의 GM 옥수 수, 3개의 GM 카놀라, 1개의 GM 면화 등 19개의 GMO에 대해 microarray를 적용한 결과 (Kim et al. 2010b)가 있으 며 이와 같이 우리나라 자체적인 기술력을 확보함으로써 보다 효율적이고 개선된 새로운 GMO 분석법 마련을 위 한 시도들이 진행되고 있다.Fig. 1) (Ikami et al. 2010). ...
Since the first commercial GM plant, the FlavrSavr tomato, authorized in 1994, more than 140 GM plants were authorized for marketing globally. For the authorization and labelling of GM plants, the detection methods for genes introduced and proteins expressed in GM plants were developed qualitatively and quantitatively. This review presented the detection methods, conventional PCR, multi-plex PCR and real-time PCR, for soybean, maize, canola and cotton as the dominant GM plants. Also, microarray assay and nanotechnology as new approaches for detection methods for GM plants were investigated.
... This technology is based on the visualization of hybridization reactions between nucleic acids in a sample with unique nucleic acid sequences of known pathogens immobilized on a solid surface. Microarrays can detect up to thousands of unique sequences, and the range of organisms detected can be expanded to include not only all the different pathogens which can affect a host plant but also specific plant features, such as the variety or the presence of GM traits (Germini et al. 2005). However, production of hybridisation signals can be highly variable and extremely sensitive to minor technical differences (Carmichael 2012). ...
The main goal of seed pathology research and practice is the production and dissemination of high-quality, disease-free seed that maximizes potential crop productivity and value. Presently, the largest part of official seed health tests requires the growth of pathogens (direct methods) but molecular biology offers new tools to diagnose fungal pathogens in/on seeds (indirect methods), reducing the time required by direct methods and improving the output of seed health tests. However, molecular methods suffer some drawbacks and require a more difficult validation procedure than direct methods. This chapter aims at describing the historical context, and the development and implementation of methods for the detection and monitoring of seed-borne plant pathogenic fungi in Europe, with special emphasis on innovative methods.
... PNA can be used on microarrays and other biosensors. PNA microarray combined with PCR could detect genetically modified organisms (GMOs) in food [20]. Every Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize was correctly identified in the tested samples. ...
... Microarray-based approaches also played an important role in GMO multidetection. Several so-called DNA chips presenting different degrees of multiplexing have been developed [12][13][14][15][16]. A method demonstrating the use of ten different padlock probes for GMO detection was described [17,18] and the recently described novel alternative method named NAIMA [19,20] is based on multiplex quantitative DNA-based target amplification coupled to microarray detection. ...
The ever-increasing production of genetically modified crops generates a demand for high-throughput DNA-based methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis by DNA based methods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.
... 768 If the probes are chemically attached on the surface, the latter must be suitable 769 for linking of specific functional groups or must be activated by introduction of 770 appropriate functional groups. Probes can be directly linked to the surface, but 771 normally a 15-25 atom spacer should be added to the tail of the probe in order 772 to allow sufficient space for hybridization, as tested in our own experience for 773 PNA microarrays [145]. Direct linking is necessary when the direct readout of 774 the hybridization is necessary, such as in carbon nanotube electrochemical devices 775 allowing the detection of single molecule target DNA [146]. ...
DNA detection can be achieved using the Watson-Crick base pairing with
oligonucleotides or oligonucleotide analogs, followed by generation of a
physical or chemical signal coupled with a transducer device. The nature
of the probe is an essential feature which determines the performances
of the sensing device. Many synthetic processes are presently available
for "molecular engineering" of DNA probes, enabling label-free and
PCR-free detection to be performed. Furthermore, many DNA analogs with
improved performances are available and are under development; locked
nucleic acids (LNA), peptide nucleic acids (PNA) and their analogs,
morpholino oligonucleotides (MO) and other modified probes have shown
improved properties of affinity and selectivity in target recognition
compared to those of simple DNA probes. The performances of these probes
in sensing devices, and the requirements for detection of unamplified
DNA will be discussed in this chapter. Chemistry and architectures for
conjugation of probes to reporter units, surfaces and nanostructures
will also be discussed. Examples of probes used in ultrasensitive
detection of unamplified DNA are listed.
... A wide range of alternatives to conventional gel electrophoresis exist for detection and identification of the PCR amplified targets: capillary gel electrophoresis (Garcia-Canas et al., 2004b;Heide et al., 2008a,b;Nadal et al., 2006), hybridisation to labelled and coloured beads and flow cytometry (Fantozzi et al., 2008), array hybridisation (Germini et al., 2005;Hamels et al., 2009;Leimanis et al., 2006;Morisset et al., 2008a;Prins et al., 2008;Xu et al., 2007Xu et al., , 2006b, immunological detection with dipsticks (Kalogianni et al., 2006), surface plasmon resonance (Feriotto et al., 2003), various electrochemical sensors (Kumar and Kang, 2007;Sun et al., 2007Sun et al., , 2008Xu et al., 2006a) and detection by liquid chromatography and mass spectrometry (Shanahan et al., 2006). ...
This paper presents an overview of GMO testing methodologies and how these have evolved and may evolve in the next decade. Challenges and limitations for the application of the test methods as well as to the interpretation of results produced with the methods are highlighted and discussed, bearing in mind the various interests and competences of the involved stakeholders. To better understand the suitability and limitations of detection methodologies the evolution of transformation processes for creation of GMOs is briefly reviewed.
... The method is demonstrated that PNA array is efficient for simultaneous detections. Its sensitivity is however affected from the length of probes and their distance from the slide surface (Germini et al., 2005). ...
Genetically modified (GM) crops are the plant varieties carrying single or multiple transgenes in their genomes modified by recombinant DNA technology. Detection of transgenic elements associated with GM crops is an important issue for their traceability in food chain and also for the risk assessments related to environment and transgene introduction into human diet. A number of methods have been developed for screening GM crop products with the aim of increasing reliability and molecular sensitivity. This review article is focused on the published methods which are mainly based on PCR and DNA hybridizations as well as biosensors as a recently utilized technology. DNA hybridization methods including probe immobilization on solid surfaces and subsequent hybridization by target DNA are variously depended on surface types, probe labeling conditions or some modifications such as the use of peptide nucleic acids (PNA). Quantitative real-time PCR (qRT-PCR) is the most routinely used and compatible method for quantification which is a crucial issue in GMO content analyses. Finally, biosensors represent more advanced assays with high detection sensitivity provided by specific transducers sense DNA hybridization events. Progress in technical implementations related to GM crop analyses will contribute not only to environmental safety but also to guarantee global market functioning and the consumer rights to choose.
... In addition, the introduction of mismatches has a stronger effect on the stability of PNA/DNA interactions in comparison to DNA/DNA duplexes, demonstrating the high specificity of PNAs [16]. Several in vitro techniques make use of the extraordinary affinity of PNAs [17,18]. In vivo techniques also strongly benefit from the highly specific binding to mRNAs, however, so far unmodified aegPNAs have not been successful in injection experiments for gene specific knock down in animal models [19]. ...
Synthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications.
We generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes.
PNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.
... As these problems become resolved it may be possible in the future to combine the detection of broader ranges of different types of pathogens from large host ranges on single arrays. It may also be possible to include the screening of other traits in plants, such as variety, presence of GM events (Germini et al., 2005;Rudi et al., 2003) or even the expression of genes caused by environmental stress that could be confused with pathogen infection (Tamaoki et al., 2004). ...
In the last decade, developments in molecular (nucleic acid-based) diagnostic methods have made significant improvements in
the detection of plant pathogens. By using methods such as the polymerase chain reaction (PCR), the range of targets that
can now be reliably diagnosed has grown to the extent that there are now extremely few, known pathogens that cannot be identified
accurately by using laboratory-based diagnostics. However, while the detection of pathogens in individual, infected samples
is becoming simpler, there are still many scenarios that present a major challenge to diagnosticians and plant pathologists.
Amongst these are the detection of pathogens in soil or viruses in their vectors, high throughput testing and the development
of generic methods, that allow samples to be simultaneously screened for large numbers of pathogens. Another major challenge
is to develop robust technologies that avoid the reliance on well-equipped central laboratories and making reliable diagnostics
available to pathologists in the field or in less-developed countries. In recent years, much of the research carried out on
phytodiagnostics has focussed in these areas and as a result many novel, routine diagnostic tests are becoming available.
This has been possible due to the introduction of new molecular technologies such real-time PCR and microarrays. These advances
have been complemented by the development of new nucleic acid extraction methods, increased automation, reliable internal
controls, assay multiplexing and generic amplification methods. With developments in new hardware, field-portable real-time
PCR is now also a reality and offers the prospect of ultra-rapid, on-site molecular diagnostics for the first time. In this
paper, the development and implementation of new diagnostic methods based upon novel molecular techniques is presented, with
specific examples given to demonstrate how these new methods can be used to overcome some long-standing problems.
Although many potential applications in early clinical diagnosis have been proposed, the use of a surface plasmon resonance imaging (SPRI) technique for non-invasive prenatal diagnostic approaches based on maternal blood analysis is confined. Here, we report a nanoparticle-enhanced SPRI strategy for a non-invasive prenatal fetal sex determination based on the detection of a Y-chromosome specific sequence (single-gene SRY) in cell-free fetal DNA from maternal plasma. The SPR assay proposed here allows for detection of male DNA in mixtures of 2.5 aM male and female genomic DNAs with no preliminary amplification of the DNA target sequence, thus establishing an analytical protocol that does not require costly, time-consuming, and prone to sample contamination PCR-based procedures. Afterward, the developed protocol was successfully applied to reveal male cell-free fetal DNA in the plasma of pregnant women at different gestational ages, including early gestational ages. This approach would pave the way for the establishment of faster and cost-effective non-invasive prenatal testing.
Genetically Modified (GM) foods are becoming the future of agriculture on surviving global natural disasters and climate change by their enhanced production efficiency and improved functional properties. On the other hand, their adverse health and environmental effects, ample evidence on transgene leakage of Genetically Modified Organisms (GMOs) to crops have raised questions on their benefits and risks. Consequently, low-cost, reliable, rapid, and practical detection of GMOs have been important. GMO-detection platforms should be capable of stably storing detection reagents for long-delivery distances with varying ambient temperatures. In this study, we developed an event-specific, closed tube colorimetric GMO detection method based on Loop-Mediated Isothermal Amplification (LAMP) technique which can be integrated into GMO-detection platforms. The entire detection process optimized to 30 minutes and isothermally at 65°C. The durability of the LAMP mixture in the test tubes showed that the LAMP reaction mixture, in which Bst polymerase and DNA sample was later included, yielded DNA amplicons for 3 days at room temperature, and for 6 days at 4°C.
•Simple, stable, and cheap storage method of LAMP reaction mixture for GMO-detection technologies.
•GMO-detection platforms can stably store detection reagents for long-delivery distances with varying ambient temperatures.
•Any DNA sample can be used in the field or resource-limited setting by untrained personnel.
A novel enzyme-labelled voltammetric magnetogenoassay for DNA sensing based on the use of carboxyl-surface coated magnetic microbeads functionalized with PNA probes and subsequent read-out on screen-printed electrode (SPE) substrates was developed. The assay was validated for determination of non-amplified genomic DNA from genetically modified Roundup Ready soy. Outstanding performance with respect to other genoassays requiring preliminary amplification of target DNA via PCR was demonstrated. The analytical performance was also improved compared to previous methods based on the immobilization of the same PNA probes on SPE substrates, since the method was found capable of achieving LOD and LOQ of 415 fM and 995 fM, respectively.
The ability of the magnetogenoassay to detect the presence of Roundup Ready soy DNA sequence was tested on genomic DNA extract from European Reference Material soy flours, demonstrating the capability of the method to match the European Union regulation for labelling of food containing a percentage of GM products greater than 0,9%.
Microbes are ubiquitously present in nature and their presence in environment or food materials is natural and unavoidable. Presence of microbes in food products may be beneficial or may cause serious threat to health upon consumption. With reference to European Commission (EC), Food and Drug Administration (FDA) and Hazard Analysis Critical Control Point (HACCP) guidelines, detection of illness causing foodborne microbes/pathogens and/or presence of GMOs has become a burning issue that needs to be attended on priority basis. This chapter is an attempt to describe various molecular techniques for detection of foodborne pathogens and GMOs. Emphasis has been made to elaborate basic to next-generation techniques developed related to PCR, microarrays, and DNA sequencing that have achieved unprecedented level of performance in terms of sensitivity, speed, precision, and accuracy.
Antibiotics have long been used for anti-infective control of bacterial infections, growth promotion in husbandry, and prophylactic protection against plant pathogens. However, their inappropriate use results in the emergence and spread of multiple drug resistance (MDR) especially among various bacterial populations, which limits further administration of conventional antibiotics. Therefore, the demand for novel anti-infective approaches against MDR diseases becomes increasing in recent years. The peptide nucleic acid (PNA)-based technology has been proposed as one of novel anti-infective and/or therapeutic strategies. By definition, PNA is an artificially synthesized nucleic acid mimic structurally similar to DNA or RNA in nature and linked one another via an unnatural pseudo-peptide backbone, rendering to its stability in diverse host conditions. It can bind DNA or RNA strands complimentarily with high affinity and sequence specificity, which induces the target-specific gene silencing by inhibiting transcription and/or translation. Based on these unique properties, PNA has been widely applied for molecular diagnosis as well as considered as a potential anti-infective agent. In this review, we discuss the general features of PNAs and their application to various bacterial pathogens as new anti-infective or antimicrobial agents.
Deoxyribonucleic acid (DNA) is a ubiquitously occurring cell constituent that provides highly specific information on organisms at every taxonomic level. This chapter outlines the basic principles, challenges and current developments in DNA-based detection of genetically modified (GM) crops. The European Commission's Reference Laboratory (EU-RL GMFF) provides official methods for the specific detection and quantification of material from different GMOs. Despite these valuable methods, major analytical challenges arise from the increasing number of authorised and unauthorised GMO, the lack of reference materials, and the need to determine GMO contents in composite and processed foods. The need to quantify the content of GMO in a product varies among different countries, depending on tolerance thresholds and labelling policies. The quantification in composed and processed products requires simultaneous assessment of the recombinant DNA and of a species- or taxon-specific reference gene.
The need for complete and truthful information about the food products we eat is a primary request from consumers. In the last years many regulations have appeared in order to avoid food adulteration, mislabelling and fraud. For these reasons, the authentication of food products must be guaranteed, and therefore accurate and reliable analytical methods are needed in order to verify that the components used in a food product are of the nature or quality declared by the seller. Many different instrumental techniques have been proposed for food authentication, and even though traditional methods are being extensively used, the use of tools for the definition of molecular targets, such as DNA-based and proteomics techniques, is helping to complement existing methodologies. In this chapter we offer a comprehensive and updated overview of these molecular techniques that have been applied to date for the authentication of food components.
Genetically modified organisms and the derived food and feed are subject to legal restrictions. A number of EU regulations determine GMOs which may appear on the market and their contents in food and feed. These restrictions require accurate and sensitive methods for identification and quantification of GM products by PCR. Moreover, the threat of unauthorized GMO import necessitates the search for new approaches and methods.
This chapter contains section titled:
In this paper we describe the recent advances in the synthesis and application of polyamide DNA analogues peptide nucleic acid (PNAs) and modified PNAs, obtained by our research group in the context of national and international projects dedicated to DNA targeting in the fields of diagnostics and drug development. Modification of the PNA backbone by inserting substituents which create stereocenters either at position C2, C5 or both have been studied. Racemization-free synthetic procedures were developed in order to preserve the optical purity of the modified chiral PNAs. The control of stereochemistry allowed a fine-tuning of the DNA binding properties in accordance with the preference of each PNA for right- or left-handedness. Modified nucleobases are also shown to be very promising for increasing DNA binding, allowing additional specific interactions with the target base. The availability of modified PNAs has provided new efficient tools for the recognition of DNA sequences with single nucleotide polymorphism (SNP), which have been applied to the detection of DNA relevant to medical diagnosis as well as to food analysis. PNA probes to identify genetically modified organisms (GMOs), allergens, and plant varieties have been developed and used in combination with several analytical techniques. Ultrasensitive detection of DNA was also achieved using PNA probes in a microfluidic platform. Moreover, PNAs and modified PNAs have been used in therapeutics and were shown to inhibit gene expression by an anti-gene mechanism, a promising strategy for the development of new, highly specific antitumor drugs.
Genetically modified organisms and the derived food and feed are subject to legal restrictions. A number of EU regulations determine GMOs which may appear on the market and their contents in food and feed. These restrictions require accurate and sensitive methods for identification and quantification of GM products by PCR. Moreover, the threat of unauthorized GMO import necessitates the search for new approaches and methods. © 2014, Czech Society of Chemical Engineering. All right reserved.
An efficient system for detection and quantitation of the Galanthus nivalis agglutinin "GNA" transgene, with reference gene Sucrose Phosphate Synthase (SPS), in GM rice, a SYBR green based real time PCR assay was optimized. Snowdrop lectin gene product(GNA) proved to be toxic towards the pest "brown plant hopper" (BPH) and green leaf hopper (GLH) feeding on rice. The transgenic rice line developed employing GNA and non transgenic control variety (Chaitanya) was used in this study. The DNA isolated from transgenic rice was serially diluted to create a standard curve of "GNA" gene and regression analysis yielded R2 value of 0.972. As low as one copy number could be detected using the developed protocol.
Introduction DNA-Based Methodologies for GMO Analysis Strategies for Detection, Identification, and Quantification of GMOs Application of Current Methodologies to GMO Analysis References
Introduction Biosensors for food control and safety Conclusions
This chapter outlines sampling, detection, identification, and quantification of genetically modified organisms (GMOs). GMOs can be defined as organisms whose genetic constitution has been modified by gene technology. Normally, a highly reliable sample for GMO testing of food commodities can only be obtained with very costly sampling plans. Sampling to test for the presence of GMOs or derived material is often compared with sampling to test for the presence of mycotoxins because the distribution of both mycotoxins and GMO-derived materials may be extremely heterogeneous and the overall contamination from the mother and father may not be the same. Purification helps concentrate the analyte so that it may be more detectable and possibly even quantifiable. This step typically removes cell debris and releases the analyte from any chemically or physically attached compounds that may otherwise interfere with the analytical reaction, usually by inhibiting detection of the analyte. The DNA-based sequence information, corresponding to the whole genome, is always complete in the nucleus of the cells of all tissues. On the contrary, only parts of the total spectrum of RNA sequences that can be produced by the plant may be found in a specific sample, because the transcription of mRNA from the DNA depends on a series of regulatory elements.
Peptide nucleic acid (PNA) is a mimic of DNA that shows a high chemical stability and can survive the enzymatic degradation of nucleases and proteases. The superior binding properties of PNA enable the formation of PNA/DNA or PNA/RNA duplex with excellent thermal stability and unique ionic strength effect. The introduction of microarray makes it possible to achieve accurate, high throughput parallel analysis of DNA or RNA with a highly integrated and low reagents consuming device. This powerful tool expands the applications of PNA in genotyping based on single nucleotide polymorphism (SNP) detection, the monitoring of disease-related miRNA expression and pathogen detection. This review paper discusses the fabrications of PNA microarrays through in situ synthesis strategy or spotting method by automatic devices, the various detection methods for the microarray-based hybridization and the current applications of PNA microarrays.
Copyright © 2014 Elsevier B.V. All rights reserved.
The feasibility of a biosensor for DNA label-free detection, based on long period fiber gratings, has been investigated. The surface of the grating has been functionalized with Peptide Nucleic Acid (PNA) probes. DNA strands, matched with the PNA probes, have been immobilized on the surface itself. The possibility of a resonant wavelength shift in the transmission spectrum due to the DNA capture will be discussed. The problem of reusing the sensor for multiple measurements will also be addressed.
Peptide nucleic acids (PNAs) are synthetic oligonucleotide analogues based on a pseudopeptide backbone that bind complementary DNA or RNA with high affinity and specificity. In this chapter, three PNA-based genotyping assays are described: PCR clamping, fluorescence-based recognition, and microarray platform. The first two methods are performed in solution, while the microarray method uses a solid surface.
Food safety and quality are very important issues receiving a lot of
attention in most countries by producers, consumers and regulatory and
control authorities. In particular, DNA analysis in food is becoming
popular not only in relation to genetically modified products (GMOs), in
which DNA modification is the "clue" of the novelty, but also in other
fields like microbiology and pathogen detection, which require long
times for the cultivation and specially in cases in which the
microorganisms are not cultivable like some viruses, as well as for
authenticity and allergen detection. A new topic concerning
"nutrigenetics and nutrigenomics" has also been mentioned, very
important but still in its infancy, which could lead in the future to a
personalized diet. In this chapter we have described the main areas of
food research and fields of application where DNA analysis is being
performed and the relative methods of detection, which are generally
based on PCR. The possibility/opportunity to detect DNA without previous
amplification (PCR-free) will be discussed. We have examined the
following areas: (1) genetically modified foods (GMOs); (2) food
allergens; (3) microbiological contaminations; (4) food authenticity;
(5) nutrigenetics/nutrigenomics.
Surface plasmon-based optical methods using peptide nucleic acids (PNAs) probes have recently emerged as powerful and highly sensitive tools for DNA and RNA detection. Surface plasmon resonance (SPR) is a powerful technique for the high sensitive and label-free detection of nucleic acids while PNAs help in targeting complementary sequences with exceptionally high selectivity and sensitivity. The present review summarizes the research efforts paid over the last years in the above mentioned field. In particular, fundamental aspects of the SPR detection of the PNA/DNA hybridization will be discussed. Then, research activities aimed at demonstrating the ultrasensitive DNA detection and the possibilities offered in single nucleotide polymorphisms detection will be presented. The contribution given by surface plasmon-allied techniques and by the combined use of the electrochemical detection will be also discussed. The SPR detection of DNA by chiral PNAs will be finally mentioned.
Surface plasmon resonance represents a new tool for the simple, fast and cheap nucleic acid detection. Large efforts have been paid during the last decade with the aim to develop even more sensitive and specific SPR-based methods to be used for the direct detection of genomic DNA. This Chapter, after a description of fundamentals of surface plasmon resonance and allied techniques, will review the state-of-the-art of recent platform and assay developments and will provide an overview of recent uses of SPR techniques for the direct detection of non-amplified nucleic acids.
Surface plasmon resonance imaging (SPRI) is a powerful tool for simple, fast and cheap nucleic acid detection. Great efforts have been made during the last decade with the aim of developing even more sensitive and specific SPRI-based methods to be used for the direct detection of DNA and RNA. Here, after a description of the fundamentals of SPRI, the state of the art of recent platform and assay developments is presented, with special attention given to advances in SPRI signal enhancement procedures.
Robust and validated analytical methods are needed for the implementation of GM-food and feed legislation. The validation, based on the characterisation of a method for its sensitivity, accuracy and precision, is performed in-house or in collaborative trials according to good laboratory practice to comply with internationally harmonised standards. The way in which this is implemented depends on the analyte, influences of the food/feed matrix and other requirements that need to be met. Certified reference materials, when matrix matches and contains the appropriate concentration of the certified analyte, are a useful tool in validation. Some acceptability criteria, associated to ad-hoc statistics (e.g. percent bias, repeatability and reproducibility) are useful for the purposes of testing and decision-making. However, the interpretation of validation results also depends on professional judgment and expertise. In such respect, fuzzy logic-based techniques (complementary to conventional statistics) are advocated to support the continuous progress of GMO science and routine laboratory analyses.
Hypersensitivity to peanut is a public health problem, since the ingestion of even low amounts of peanut can trigger severe
allergic reactions. Allergic consumers rely on the information provided on the label of foodstuffs to identify products that
might endanger their health. In order to protect the allergic consumer methods are required for the detection of allergenic
ingredients. For this purpose we have developed three real-time polymerase chain reaction (PCR) assays, based on TaqMan chemistry,
that are capable of detecting peanut specific DNA sequences in food products. The peanut specific sequence targeted for detection
is located within the gene family of the allergen Ara h 3. The occurrence of multiple Ara h 3 sequences in the peanut genome
increases the chance to achieve a good sensitivity. DNA extraction is also known to affect detection by PCR, therefore the
efficiency of several different DNA extraction methods was compared. The methods reported here are capable of detecting 2.5pg
peanut DNA (less than one copy of peanut genome equivalent) and all three assays were successfully applied to detect peanut
traces in a model food product where they could detect 10mgkg−1 peanut.
The soybean cultivar Yumeminori, which lacks the α′- and α-subunits of β-conglycinin, carries both naturally occurring and induced mutations. While the cause of the natural mutation resulting in the α′-subunit deficiency has been determined, the induced mutation in the CG-2 gene encoding the α-subunit has not been characterized at the molecular level. In this study, we identified a four base pair insertion in the first exon of CG-2, which introduced a premature stop codon. The insertion co-segregated with the lack of α-subunit, indicating that this mutation is the cause of the α-subunit deficiency. A multiplex PCR method of testing for the presence or absence of α′- and α-subunits was developed based on the sequences of mutated and wild-type alleles. This PCR-based test was also capable of detecting the presence of wild-type genes when Yumeminori DNA samples were contaminated with wild-type DNA at levels of 0.2% or greater. Thus, this method will be useful both for marker-assisted selection in soybean breeding programs, and for seed purity tests in food industries.
A method using polymerase chain reaction (PCR) was designed for the detection of genetically modified maize (GM-maize). There are four lines of GM-maize imported from the United States, and the presence of recombinant deoxyribonucleic acid (DNA) in the maize could be detected with four pairs of specific oligonucleotide primers designed from the sequences of the newly introduced genes. The maize zein gene was also detected as an internal control. This method allows specific detection of each of Bt11, Event176, MON810 and LIBERTY by using pairs of specific primers designed to amplify a segment including part: of the exogenously introduced sequence and part of the intrinsic maize sequence. The detection sensitivity was about 0.05% for Event176, MON810 and LIBERTY, and about 0.01% for Bt11. To distinguish among three insect-resistant GM-maize lines, we designed a multiplex PCR method. These three GM-maize lines were distinguishable on the basis of the expected lengths of their amplicons.
Numerous analytical methods, both qualitative and quantitative, have been developed to determine reliably the presence and/or the amount of genetically modified organisms (GMOs) in agricultural commodities, in raw agricultural materials and in processed and refined ingredients. In addition to the "classical" methods for DNA and protein analysis, e.g. polymerase chain reaction and enzyme linked immunosorbent analysis, certain types of GMO-containing matrices can be profiled by complementary chemical analysis methods such as chromatography and near infrared spectroscopy. This review summarises the status of the most widely used GMO analysis technologies, identifies new areas of analytical investigation and discusses current needs and future challenges.
Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device originally designed for peptide synthesis. At approximately 96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use, because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched versions was possible at salt concentrations down to 10 mM Na+and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.
Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.
The abbreviated name, ‘mfold web server’, describes a number of closely related software applications available on the World
Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server
is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making
use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this
software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency
plots and ‘energy dot plots’, are available for the folding of single sequences. A variety of ‘bulk’ servers give less information,
but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold. This URL will be referred to as ‘MFOLDROOT’.
We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5' 'HEAD' region and a 3' region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1-2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5' nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection.
Peptide Nucleic Acid (PNA) is conceptually a DNA analog, but in a chemical sense PNA bridges peptides and nucleic acids by virtue of its pseudopeptide backbone and its nucleobases. Since this mimic was introduced five years ago, convenient synthetic routes to PNA monomers as well as oligomers have been devised, and a number of structural modifications have been introduced into PNA. In addition, extensive research has shed light on the interesting properties of PNA, demonstrating the development potential of PNA into biomolecular tools, as well as antisense and/or antigene drugs. This review focuses primarily on the chemical aspects of PNA and its modifications, including the introduction of chirality, the elongation of the pseudopeptide backbone as well as the permutation of functional groups within the backbone, and the introduction of a non-natural nucleobase. In addition, the effects of covalently linking two PNA oligomers will be described. Furthermore, the ability of PNA to hybridize to DNA or RNA targets with a high sequence specificity will be discussed, as will the secondary structure of such hybrids and that of PNA itself. Finally, (potential) applications of PNA are briefly dealt with herein.
Arrays of up to some 1000 PNA oligomers of individual sequence were synthesised on polymer membranes using a robotic device
originally designed for peptide synthesis. At ∼96%, the stepwise synthesis efficiency was comparable to standard PNA synthesis
procedures. Optionally, the individual, fully deprotected PNA oligomers could be removed from the support for further use,
because an enzymatically cleavable but otherwise stable linker was used. Since PNA arrays could form powerful tools for hybridisation
based DNA screening assays due to some favourable features of the PNA molecules, the hybridisation behaviour of DNA probes
to PNA arrays was investigated for a precise understanding of PNA-DNA interactions on solid support. Hybridisation followed
the Watson-Crick base pairing rules with higher duplex stabilities than on corresponding DNA oligonucleotide sensors. Both
the affinity and specificity of DNA hybridisation to the PNA oligomers depended on the hybridisation conditions more than
expected. Successful discrimination between hybridisation to full complementary PNA sequences and truncated or mismatched
versions was possible at salt concentrations down to 10 mM Na+ and below, although an increasing tendency to unspecific DNA binding and few strong mismatch hybridisation events were observed.
Peptide nucleic acid (PNA), originally developed as a gene-targeting drug, has demonstrated remarkable hybridization properties towards complementary oligonucleotides. Biosensors based on replacement of the DNA recognition layer with a PNA one, offer greatly improved distinction between closely related sequences, as well as several other attractive advantages. The present review discusses the unique structural, hybridization, and recognition features of PNA probes, along with the opportunities accrued from the use of PNA recognition layers in DNA diagnostics.
PCR methods for the detection of genetically modified organisms (GMOs) were developed that can be used for screening purposes and for specific detection of glyphosate-tolerant soybean and insect-resistant maize in food. Primers were designed to amplify parts of the 35S promoter derived from Cauliflower Mosaic Virus, the NOS terminator derived from Agrobacterium tumefaciens and the antibiotic marker gene NPTII (neomycin-phosphotransferase II), to allow for general screening of foods. PCR/hybridization protocols were established for the detection of glyphosate-tolerant RoundUp Ready soybean and insect-resistant Bt-maize. Besides hybridization, confirmation of the results using restriction analysis was also possible. The described methods enabled a highly sensitive and specific detection of GMOs and thus provide a useful tool for routine analysis of raw and processed food products. Keywords: Polymerase Chain Reaction; PCR; genetically modified organisms; food analysis
A cloth-based hybridization array system (CHAS) was developed for detection of amplicons generated in a multiplex PCR targeting transgenic 35S and NOS sequences, as well as corn invertase and soy lectin genes. The CHAS provided confirmation of each amplicon on the basis of hybridization with specific capture probes, and enabled ease of discrimination of the multiplex PCR products by visualization of the amplicons on the array.
Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0–0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA–DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA–DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA–DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.
The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products.Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil.If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker.Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity.Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers.
The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) containing all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate constants for association (k(a)) and dissociation (k(d)) of the duplex formation as well as the thermal stability (melting temperature, T(m)) of the duplexes have been determined. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences containing single mismatches at various positions in the center resulted in increased dissociation and decreased association rate constants. T(m) values for PNA x RNA duplexes are on average 4 degrees C higher than for PNA x DNA duplexes and follow quantitatively the same variation with mismatches as do the PNA x DNA duplexes. Also a faster k(a) and a slower k(d) are found for PNA x RNA duplexes compared to the PNA x DNA duplexes. An overall fair correlation between T(m), k(a), and k(d) is found for a series of PNA x DNA and PNA x RNA duplexes although the determination of k(a) seemed to be prone to artifacts of the method and was not considered capable of providing absolute values representing the association rate constant in bulk solution.
Peptide nucleic acid (PNA), originally developed as a gene-targeting drug, has demonstrated remarkable hybridization properties towards complementary oligonucleotides. Biosensors based on replacement of the DNA recognition layer with a PNA one, offer greatly improved distinction between closely related sequences, as well as several other attractive advantages. The present review discusses the unique structural, hybridization, and recognition features of PNA probes, along with the opportunities accrued from the use of PNA recognition layers in DNA diagnostics.
Several exciting new developments in the applications of the DNA mimic peptide nucleic acid (PNA) have been published recently. A possible breakthrough may have come in efforts to develop PNA into gene therapeutic drugs. In eukaryotic systems, antisense activity of PNAs (as peptide conjugates) has been reported in nerve cells and even in rats upon injection into the brain, and antisense activity has also been demonstrated in Escherichia coli. PNA hybridization technology has developed rapidly within in situ hybridization, and exciting new methods based on MALDI-TOF detection have also been presented.
Within the past couple of years peptide nucleic acid (PNA) antisense and antigene technology has entered the realm of biological and preclinical studies. This is primarily due to the development of a number of novel methods for more efficient delivery of PNA oligomers to eukaryotic cells. These methods have allowed ex vivo studies on cells in culture to be performed, and parallel in vivo studies are also slowly emerging. Although many issues still need to be resolved and several of the most recent results cannot be rationalized in a straight forward manner by existing knowledge, the immediate future should supply a more solid foundation for assessing the prospects of PNA antisense and antigene technology both in the context of functional genomics and medicine.
Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. In this article, protein- and DNA-based methods employing western blots, enzyme-linked immunosorbant assay, lateral flow strips, Southern blots, qualitative-, quantitative-, real-time- and limiting dilution-PCR methods, are discussed. Where information on modified gene sequences is not available, new approaches, such as near-infrared spectrometry, might tackle the problem of detection of non-approved genetically modified (GM) foods. The efficiency of screening, identification and confirmation strategies should be examined with respect to false-positive rates, disappearance of marker genes, increased use of specific regulator sequences and the increasing number of GM foods.
We report here a preliminary evaluation of a microfabricated disposable-type peptide nucleic acid (PNA) array, with a 20-channel electrode, for the detection of cancer gene c-Ki-ras. Synthetic 15-mer PNA probes complimentary to ras sequence modified with cysteine were immobilized on the gold electrodes on the array. The electrochemical PNA array was reacted with 20-mer oligonucleotide target or 128 bp PCR product for 1 h. The anodic current derived from an electrochemically active DNA binder Hoechst 33258 was measured using the PNA array in the 50 microL of reaction chamber. The anodic current from Hoechst 33258 increased with increasing the concentration of PCR product of ras gene in the range from 10(11) to 10(15) copy mL(-1). The single base mismatch mutations of c-Ki-ras/61 were also detected using the electrochemical PNA array.
The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.
Peptide nucleic acid (PNA) microarrays for the detection of Roundup Ready soybeans in food have been prepared. PNA probes are known to be more efficient and selective in binding DNA sequences than the analogous oligonucleotides and are very suitable to be used for diagnostics in food. PNAs of different lengths were carefully designed and synthesized by solid-phase synthesis on an automatic synthesizer adopting the BOC strategy. PNAs were purified by HPLC and characterized by HPLC/MS. The probes were spotted on a functionalized surface to produce a microarray to be hybridized with PCR products. DNA extracted from reference material was amplified using Cy3- and Cy5-labeled primers, and the fluorescent PCR products obtained were hybridized on the microarray. Two protocols were adopted: the hybridization with dsDNA or with ssDNA obtained by digestion with the enzyme lambda exonuclease. The best results were obtained using a 15-mer PNA probe in combination with the ssPCR product derived from enzymatic digestion. The method was applied to the analysis of a sample of certified transgenic soybean flour.
In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code
microarray for the detection of hepatocyte nuclear factor-1α (HNF-1α) mutations that cause type 3 maturity onset diabetes
of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified
PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR
products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with
3′ complementarity to the specific mutation site and 5′ complementarity to the respective PNA zip-code sequence on the microarray.
The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and
the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a
much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation
sites in exon 2 of HNF-1α with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few
successful applications of PNA in DNA chip technology.
We recently developed a multiplex polymerase chain reaction (PCR) system for the simultaneous detection of four transgenic maize (MON810, Bt176, Bt11, and GA21), one transgenic soybean (Roundup Ready), and two control genes (lectin and zein). Because PCR can lead to ambiguous interpretations due to low specificity, we have developed the ligation detection reaction (LDR) combined with a universal array as a molecular tool to confirm results of PCR analysis. Here, we describe the PCR-LDR-universal array procedure and demonstrate its specificity in revealing the presence of transgenic DNA in experimental samples, raw materials, and commercial foodstuffs.
Diagnosis of HNF-1R mutations on a PNA zip-code microarray by single base extension. Nucleic Acid Res), e19. (19) Immunochemical and molecular methods. Swiss Food Manual
- J Y Song
- H G Park
- S O Jung
- J C Park
Song, J. Y.; Park, H. G.; Jung, S. O.; Park, J. C. Diagnosis of HNF-1R mutations on a PNA zip-code microarray by single base extension. Nucleic Acid Res. 2005, 33 (2), e19. (19) Immunochemical and molecular methods. Swiss Food Manual; 2001; Chapter 52b. CD-ROM 311. 510, BBL-EDMZ 3003 Bern, Switzerland.
PCR coupled with PNA-HPLC for the sensitive detection of traces of potentially allergenic hazelnut in foodstuff
- A Germini
- E Scaravelli
- F Lesignoli
- S Sforza
- R Corradini
- R Marchelli
Germini, A.; Scaravelli, E.; Lesignoli, F.; Sforza, S.; Corradini,
R.; Marchelli, R. PCR coupled with PNA-HPLC for the sensitive
detection of traces of potentially allergenic hazelnut in foodstuff.
Eur. Food Res. Technol. In press.