Paper-based device providing visual genetic signatures for precision medicine: application to breast cancer.

Genome-wide association studies have demonstrated that combinations of single nucleotide polymorphisms (SNPs), rather than individual SNPs, represent genetic signatures that correlate with heterogeneous and complex diseases. In this context, we developed a paper-based device that provides visual detection of a 10-SNP panel as a genetic signature associated with the risk for breast cancer. The method involves multiplex PCR amplification, multiplex extension reaction of allele-specific primers, without prior purification of the amplified sequences, and, finally, capture and visualization of the extension products within minutes on the device. Detection and monitoring are accomplished either by naked eye or by scanning with a common flatbed scanner. The total assay time is ∼ 2 h. The method was evaluated by using 21 clinical samples of known genotypes. The results were fully concordant with the reference method (sequencing). The proposed method is accurate, simple, rapid, and cost-effective. Visual detection does not require specialized instrumentation or highly trained technical personnel. We anticipate that the proposed device will become a useful analytical tool for precision medicine of breast cancer.

Multianalyte quantitative competitive PCR on optically encoded microspheres for an eight-gene panel related to prostate cancer.

Nucleic acid-based tests have a profound impact in every medical discipline. Because multigene tests offer higher diagnostic accuracy and lower overall cost than single assays, they are especially useful for diseases, like prostate cancer, that present variability at the molecular level and diversity of available therapeutic interventions. We have developed a quantitative competitive PCR for an eight-gene panel, related to prostate cancer, that includes five genes of the human tissue kallikrein family (KLKs), prostate-specific membrane antigen (PSMA), prostate cancer antigen 3 (PCA3), and HPRT1 as a reference gene. Using PCR as a synthetic tool, a competitor was prepared for each target sequence containing the same primer binding sites as the target but differing in a short segment to enable discrimination by hybridization. The assay involves multiplex amplification of targets and competitors followed by a multiplex hybridization assay for the 16 amplification products. The assay was performed on optically encoded microspheres with oligonucleotide probes attached to their surface. The microspheres were analyzed rapidly (1 min) by flow cytometry. The signal ratio of the target and cognate competitor is a function of the target copy number in the sample prior to amplification. The multiplexing potential of the proposed method is much higher than real-time PCR and other end-point methods since there are 100 sets of commercially available microspheres.

Τwo-panel molecular testing for genetic predisposition for thrombosis using multi-allele visual biosensors.

Thrombosis is considered as the most typical example of multigenic/multifactorial disorder. The three most common genetic risk factors for thrombotic disorders are the G1691A mutation in factor V gene (FV Leiden), the G20210Α mutation in prothrombin gene (FII), and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. An additional panel of biomarkers predisposing for thrombotic events includes the H1299R variant in factor V gene (HR2), A1298C variant in MTHFR gene, the V34L mutation in fibrinogen stabilizing factor XIII (FXIII) gene as well as the 4G/5G polymorphism in plasminogen activator inhibitor type-1 (PAI-1) gene. In this context, we report a novel, rapid and low-cost two-panel diagnostic platform for the simultaneous visual genotyping of the seven mutations (14 alleles). The proposed method comprises the following: (a) a multiplex PCR using genomic DNA isolated from peripheral blood, (b) a multiplex genotyping reaction based on allele-specific primer extension, and (c) visual detection of the genotyping reaction products by means of a multi-allele dipstick-type DNA biosensor, using gold nanoparticles as reporters. The method was applied to 40, previously characterized, and 15 blind clinical samples and the results were 100 % accurate. The proposed assay is simple to perform, requires no specialized and costly equipment, and eliminates multiple pipetting, incubation, and washing steps.

Digital camera and smartphone as detectors in paper-based chemiluminometric genotyping of single nucleotide polymorphisms.

Chemi(bio)luminometric assays have contributed greatly to various areas of nucleic acid analysis due to their simplicity and detectability. In this work, we present the development of chemiluminometric genotyping methods in which (a) detection is performed by using either a conventional digital camera (at ambient temperature) or a smartphone and (b) a lateral flow assay configuration is employed for even higher simplicity and suitability for point of care or field testing. The genotyping of the C677T single nucleotide polymorphism (SNP) of methylenetetrahydropholate reductase (MTHFR) gene is chosen as a model. The interrogated DNA sequence is amplified by polymerase chain reaction (PCR) followed by a primer extension reaction. The reaction products are captured through hybridization on the sensing areas (spots) of the strip. Streptavidin-horseradish peroxidase conjugate is used as a reporter along with a chemiluminogenic substrate. Detection of the emerging chemiluminescence from the sensing areas of the strip is achieved by digital camera or smartphone. For this purpose, we constructed a 3D-printed smartphone attachment that houses inexpensive lenses and converts the smartphone into a portable chemiluminescence imager. The device enables spatial discrimination of the two alleles of a SNP in a single shot by imaging of the strip, thus avoiding the need of dual labeling. The method was applied successfully to genotyping of real clinical samples. Graphical abstract Paper-based genotyping assays using digital camera and smartphone as detectors.

Screening non-deletion α-thalassaemia mutations in the HBA1 and HBA2 genes by high-resolution melting analysis.

BACKGROUND: Screening for "non-deletion" α-chain haemoglobin variants resulting from point mutations or short deletions/insertions has attracted an increased interest during recent years, especially in areas where α-thalassaemia is prevalent. We describe a method utilising high resolution melting analysis for detecting the 13 most common "non-deletion" α-thalassaemia mutations in populations around the Mediterranean and Middle East. METHODS: The method comprises: (1) amplification of a 1087 bp fragment for each of the duplicated α-globin genes (HBA1 and HBA2) flanking all 13 mutations using a common forward primer and different reverse primers specific for HBA1 and HBA2, respectively; (2) nested amplification of three fragments in HBA2 flanking 10 mutations and two fragments in HBA1 flanking 5 mutations; (3) High resolution melting analysis of the amplicons using a LightScanner Instrument and LC Green. RESULTS: All 13 "non-deletion" α-chain haemoglobin variants were successfully detected by high resolution melting analysis. All heterozygote samples and eight out of 10 available homozygotes were clearly differentiated from each other and from wild type in the same amplicon. Although not all homozygote samples were distinguishable from wild type samples, this should not present a problem in a clinical setting since all DNA results should be evaluated alongside the haematological and (if relevant) clinical findings in each case. CONCLUSIONS: The 13 "non-deletion" α-chain haemoglobin variants were successfully genotyped by high resolution melting analysis using LightScanner instrument and LCGreen Plus saturating dye. High resolution melting analysis is an accurate mutation scanning tool, advantageous as a closed-tube method, involving no post-PCR manipulations and requiring only around 5 min post-PCR analysis.

Absolute quantification of the alleles in somatic point mutations by bioluminometric methods based on competitive polymerase chain reaction in the presence of a locked nucleic acid blocker or an allele-specific primer.

In somatic (acquired) point mutations, the challenge is to quantify minute amounts of the mutant allele in the presence of a large excess of the normal allele that differs only in a single base pair. We report two bioluminometric methods that enable absolute quantification of the alleles. The first method exploits the ability of a locked nucleic acid (LNA) oligonucleotide to bind to and inhibit effectively the polymerase chain reaction (PCR) amplification of the normal allele while the amplification of the mutant allele remains unaffected. The second method employs allele-specific PCR primers, thereby allowing the amplification of the corresponding allele only. DNA internal standards (competitors) are added to the PCR mixture to compensate for any sample-to-sample variation in the amplification efficiency. The amplification products from the two alleles and the internal standards are quantified by a microtiter well-based bioluminometric hybridization assay using the photoprotein aequorin as a reporter. The methods allow absolute quantification of less than 300 copies of the mutant allele even in samples containing less than 1% of the mutant allele.

A nanoparticle-based sensor for visual detection of multiple mutations.

Disposable dipstick-type DNA biosensors in the form of lateral flow strips are particularly useful for genotyping in a small laboratory or for field testing due to their simplicity, low cost and portability. Their unique advantage is that they enable visual detection in minutes without the use of instruments. In addition, the dry-reagent format minimizes the pipetting, incubation and washing steps. In this work, we significantly enhance the multiplexing capabilities of lateral flow strip biosensors without compromising their simplicity. Multiplex genotyping is carried out by polymerase chain reaction (PCR) followed by a single primer extension reaction for all target alleles, in which a primer is extended and biotin is incorporated only if it is perfectly complementary to the target. Multiallele detection is achieved by multiple test spots on the membrane of the sensor, each comprising a suspension of polystyrene microspheres functionalized with capture probes. The products of the primer extension reaction hybridize, through specific sequence tags, to the capture probes and are visualized by using antibiotin-conjugated gold nanoparticles. This design enables accommodation of multiple spots in a small area because the microspheres are trapped in the fibres of the membrane and remain fixed in site without any diffusion. Furthermore, the detectability is improved because the hybrids are exposed on the surface of the trapped microspheres rather than inside the pores of the membrane. We demonstrate the specificity and performance of the biosensor for multiallele genotyping.

Carbon nano-strings as reporters in lateral flow devices for DNA sensing by hybridization.

Presently, there is a growing interest in the development of lateral flow devices for nucleic acid analysis that enable visual detection of the target sequence (analyte) while eliminating several steps required for pipetting, incubation, and washing out the excess of reactants. In this paper, we present, for the first time, lateral flow tests exploiting oligonucleotide-functionalized and antibody-functionalized carbon nanoparticles (carbon nano-strings, CBNS) as reporters that enable confirmation of the target DNA sequence by hybridization. The CBNS reporters were applied to (a) the detection of PCR products and (b) visual genotyping of single nucleotide polymorphisms in human genomic DNA. Biotinylated PCR product was hybridized with a dA-tailed probe. In one assay configuration, the hybrid is captured at the test zone of the strip by immobilized streptavidin and detected by (dT)(30)-CBNS. In a second configuration, the hybrids are captured from immobilized (dA) strands and detected by antibiotin-CBNS. As low as 2.5 fmol of amplified DNA can be detected. For visual genotyping, allele-specific primers with a 5' oligo(dA) segment are extended by DNA polymerase with a concomitant incorporation of biotin moieties. Extension products are detected either by (dT)(30)-CBNS or by antibiotin-CBNS. Only three cycles of extension reaction are sufficient for detection. No purification of the PCR products or the extension product is required.

Visual screening for JAK2V617F mutation by a disposable dipstick.

During the last 5 years, it was discovered that the JAK2V617F somatic mutation is present in virtually all patients with polycythemia vera and a large proportion of patients with essential thrombocythemia, primary myelofibrosis, and refractory anemia with ring sideroblasts and thrombocytosis. As a result, JAK2V617F was incorporated as a new clonal marker in the 2008 revision of the WHO diagnostic criteria. Current methods for JAK2 genotyping include direct sequencing, pyrosequencing, allele-specific PCR with electrophoresis, restriction fragment length polymorphism, real-time PCR, DNA-melting curve analysis, and denaturing HPLC. Some of these methods are labor intensive and time consuming, while the others require specialized costly equipment and reagents. We report a method for direct detection of the JAK2V617F allele by the naked eye using a dipstick test in a dry-reagent format. The method comprises a triprimer PCR combined with visual detection of the products within minutes by the dipstick test. Specialized instrumentation is not involved. The requirements for highly qualified technical personnel are minimized. Because the detection reagents exist in dry form on the dipstick, there is no need for multiple pipetting and incubation steps.

Peripheral alpha-synuclein levels in patients with genetic and non-genetic forms of Parkinson's disease.

BACKGROUND: Variations of α-synuclein levels have been reported in serum and plasma in Parkinson's Disease (PD) Patients. METHODS: Serum and plasma were obtained from PD patients without known mutations (GU-PD, n = 124)), carriers of the A53T/G209A point mutation in the α-synuclein gene (SNCA) (n = 29), and respective age-/sex-matched controls. Levels of total α-synuclein were assessed using an in-house ELISA assay. RESULTS: A statistically significant increase of α-synuclein levels was found in serum, but not plasma, from GU-PD patients compared to healthy controls. A statistically significant decrease of α-synuclein levels was found in serum and plasma from symptomatic A53T mutation carriers compared to healthy controls. Plasma α-synuclein levels were modestly negatively correlated with UPDRS part III score and disease duration in A53T-PD patients. CONCLUSION: Increased α-synuclein levels in serum of GU-PD patients suggest a systemic deregulation of α-synuclein homeostasis in PD. The opposite results in A53T-PD highlight the complexity of α-synuclein homeostatic regulation in PD, and suggest the possibility of reduced expression of the mutant allele.

Identification of single-nucleotide polymorphisms by the oligonucleotide ligation reaction: a DNA biosensor for simultaneous visual detection of both alleles.

Although single nucleotide polymorphisms (SNPs) can be identified by direct hybridization with allele-specific oligonucleotide probes, enzyme-based genotyping methods offer much higher specificity and robustness. Among enzymatic methods, the oligonucleotide ligation reaction (OLR) offers the highest specificity for allele discrimination because two hybridization events are required for ligation. We report the development of a DNA biosensor that offers significant advantages over currently available methods for detection of OLR products: It allows simultaneous visual discrimination of both alleles using a single ligation reaction. Detection is complete within minutes without the need for any specialized instruments. It does not involve multiple cycles of incubation and washing. The dry-reagent format minimizes the pipetting steps. The need for qualified personnel is much lower than current methods. The principle of the assay is as follows: Following PCR amplification, a single OLR is performed using a biotinylated common probe and two allele-specific probes labeled with the haptens digoxigenin and fluorescein. Ligation products corresponding to the normal and mutant allele are double-labeled with biotin and either digoxigenin or fluorescein, respectively. The products are captured by antidigoxigenin or antifluorescein antibodies, or both, that are immobilized at the two test zones of the biosensor and react with antibiotin-functionalized gold nanoparticle reporters. The excess nanoparticles bind to biotinylated albumin that is immobilized at the control zone of the biosensor. The genotype is assigned by the characteristic red lines that appear at the two test zones. The proposed DNA biosensor constitutes a significant step toward point-of-care SNP genotyping.

Bioluminometric assay for relative quantification of mutant allele burden: application to the oncogenic somatic point mutation JAK2 V617F.

Unlike the inherited mutations, which are present in all cells, somatic (acquired) mutations occur only in certain cells of the body and, quite often, are oncogenic. Quantification of mutant allele burden (percentage of the mutant allele) is critical for diagnosis, monitoring of therapy, and detection of minimal residual disease. With point mutations, the challenge is to quantify the mutant allele while discriminating from a large excess of the normal allele that differs in a single base-pair. To this end, we report the first bioluminometric assay for quantification of the allele burden and its application to JAK2 V617F somatic point mutation, which is a recently (2005) discovered molecular marker for myeloproliferative neoplasms. The method is performed in microtiter wells and involves a single PCR, for amplification of both alleles, followed by primer extension reactions with allele-specific primers. The products are captured in microtiter wells and detected by oligo(dT)-conjugated photoprotein aequorin. The photoprotein is measured within seconds by simply adding Ca(2+). We have demonstrated that the percent (%) luminescence signal due to the mutant allele is linearly related to the allele burden. As low as 0.85% of mutant allele can be detected and the linearity extends to 100%. The assay is complete within 50 min after the amplification step.

High-throughput chemiluminometric genotyping of single nucleotide polymorphisms of histamine, serotonin, and adrenergic receptor genes.

Several pharmacogenetic studies are focused on the investigation of the relation between the efficacy of various antipsychotic agents (e.g., clozapine) and the genetic profile of the patient with an emphasis on genes that code for neurotransmitter receptors such as histamine, serotonin, and adrenergic receptors. We report a high-throughput method for genotyping of single nucleotide polymorphisms (SNPs) within the genes of histamine H2 receptor (HRH2), serotonin receptor (HTR2A1 and HTR2A2), and beta(3) adrenergic receptor (ADRB3). The method combines the high specificity of allele discrimination by oligonucleotide ligation reaction (OLR) and the superior sensitivity and simplicity of chemiluminometric detection in a microtiter well assay configuration. The genomic region that spans the locus of interest is first amplified by polymerase chain reaction (PCR). Subsequently, an oligonucleotide ligation reaction is performed using a biotinylated common probe and two allele-specific probes that are labeled at the 3' end with digoxigenin and fluorescein. The ligation products are immobilized in polystyrene wells via biotin-streptavidin interaction, and the hybrids are denatured. Detection is accomplished by the addition of alkaline phosphatase-conjugated anti-digoxigenin or anti-fluorescein antibodies in combination with a chemiluminogenic substrate. The ratio of the luminescence signals obtained from digoxigenin and fluorescein indicates the genotype of the sample. The method was applied successfully to the genotyping of 23 blood samples for all four SNPs. The results were in concordance with both PCR-restriction fragment length polymorphism analysis and sequencing.

Quadruple-allele chemiluminometric assay for simultaneous genotyping of two single-nucleotide polymorphisms.

We developed a rapid, simple, cost-effective and high sample-throughput method for the simultaneous detection of four alleles in single-nucleotide polymorphisms (SNPs). The method was applied to the simultaneous genotyping of two common SNPs within the TLR4 gene, the A896G and C1196T polymorphisms. The method consists of a single PCR of the region spanning the A896G and C1196T polymorphic sites, followed by a quadruple primer extension (PEXT) reaction in a single tube. A biotinylated nucleotide is incorporated in the extended primer. All four products are captured in streptavidin (SA)-coated microtiter wells and detected with a combination of four reporters, the photoprotein aequorin (AEQ) and the enzymes alkaline phosphatase (ALP), beta-galactosidase (GAL) and horseradish peroxidase (HRP). For each SNP, 46 individuals were genotyped. The accuracy of this method was confirmed by sequencing. The proposed quadruple-allele chemiluminometric assay provides an accurate, simple, rapid, reproducible and cost-effective method for high sample-throughput genotyping of single-nucleotide polymorphisms.

Dry-reagent disposable dipstick test for visual screening of seven leukemia-related chromosomal translocations.

We report the first dry-reagent, disposable, dipstick test for molecular screening of seven chromosomal translocations associated with acute and chronic leukemia. The dipstick assay offers about 10 times higher detectability than agarose gel electrophoresis and, contrary to electrophoresis, allows confirmation of the sequence of the polymerase chain reaction (PCR) product by hybridization within a few minutes without the need of instrumentation. Biotinylated amplified DNA is hybridized with a dA-tailed probe and applied to the strip, which contains oligo(dT)-conjugated gold nanoparticles in dry form. Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line. The excess nanoparticles are captured by oligo(dA) strands immobilized at the control zone of the strip producing a second red line. We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively. A single K562 cell was detectable amidst 10(6) normal leukocytes. A dipstick test was developed for actin, as a reference gene. The dipstick assay with appropriate probes can be used for identification of the fusion transcripts involved in the translocation.

Dry-reagent disposable biosensor for visual genotyping of single nucleotide polymorphisms by oligonucleotide ligation reaction: application to pharmacogenetic analysis.

Most genotyping methods for known single-nucleotide polymorphisms (SNPs) are based on hybridization with allele-specific probes, oligonucleotide ligation reaction (OLR), primer extension or invasive cleavage. OLR offers superior specificity because it involves two recognition events; namely, the hybridization of an allele-specific probe and a common probe to adjacent positions on target DNA. OLR products can be detected by microtiter well-based colorimetric, time-resolved fluorimetric or chemiluminometric assays, electrophoresis, microarrays, microspheres, and homogeneous fluorimetric or colorimetric assays. We have developed a simple, robust, and low-cost disposable biosensor in dry-reagent format, which allows visual genotyping with no need for instrumentation. The OLR mixture contains a biotinylated common probe and an allele-specific probe with a (dA)(20) segment at the 3'-end. OLR products are denatured and applied to the biosensor next to gold nanoparticles that are decorated with oligo(dT) strands. The sensor is immersed in the appropriate buffer and all components migrate by capillary action. The OLR product is captured by immobilized streptavidin at the test zone (TZ) of the sensor and hybridizes with the oligo(dT) strands of the nanoparticles. A characteristic red line is generated due to the accumulation of nanoparticles. The excess nanoparticles are captured by immobilized oligo(dA) at the control zone of the strip, giving a second red line. We have applied successfully the proposed OLR-dipstick assay to the genotyping of four SNPs in the drug-metabolizing enzyme genes CYP2D6 ((*)3 and (*)4) and CYP2C19 ((*)2 and (*)3). The results were in agreement with direct sequencing.

High-throughput microtiter well-based bioluminometric genotyping of two single-nucleotide polymorphisms in the toll-like receptor-4 gene.

Toll-like receptors (TLRs) play a fundamental role in pathogen recognition and activation of innate immunity. Genetic variations in TLR have been associated with reduced host immune response to TLR ligands. We developed a rapid, simple and cost-effective method for identification of two common single-nucleotide polymorphisms (SNPs) within TLR4 gene in a high-throughput format. The method consists of a single polymerase chain reaction of the region spanning the A896G and C1196T polymorphic sites, followed by two primer extension reactions at each site using primers that carry a (dA)(24) segment at the 5' end. A biotinylated nucleotide is incorporated in the extended primer. The products are captured in microtiter wells coated with streptavidin and detected using a (dT)(30)-conjugated photoprotein aequorin. A total of 209 individuals were genotyped for each SNP. The A896G and C1196T polymorphisms were found to be in linkage disequilibrium; 186 individuals (89%) were wild-type homozygous (A/A or C/C), 22 (10.5%) were heterozygotes (A/G or C/T), and 1 (0.5%) was homozygous for the mutation (G/G or T/T). The accuracy of this method was confirmed by sequencing. The newly developed method may be useful for association studies of these two SNPs with several diseases.

Advances in molecular techniques for the detection and quantification of genetically modified organisms.

Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.

Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension-application to the variants of mannose-binding lectin gene.

Mannose-binding lectin (MBL) is a key component of the innate immune system, and its deficiency is associated with increased susceptibility to various infections and autoimmune disorders. Since several nucleotide variations in the mannose-binding lectin 2 gene (MBL2) have been associated with the functional deficiency of MBL, there is a growing need to screen its allelic variants and develop genotyping methods for MBL2. In this context we propose a rapid, robust, cost-efficient, and automatable method for detecting all known allelic variants of MBL2. This report introduces for the first time the photoprotein aequorin as a reporter in genotyping by primer extension (PEXT) reactions. The method involves a single PCR amplification of a genomic region that spans all six variant nucleotide sites, i.e., three structural mutations in exon 1 (c.154C>T, pArg52Cys; c.161A>G, p.Gly54Asp; and c.170A>G, p.Gly57Glu), two single nucleotide polymorphisms (SNPs) at positions c.-619G>C and c.-290G>C (promoter region), and one SNP at position c.-66C>T of the 5' untranslated region. PCR is followed by PEXT reactions for each site. Biotin-dUTP is incorporated in the extended primer. The genotyping primers contain a poly(dA) segment at their 5' end. The products are captured by hybridization on the surface of microtiter wells that are coated with a poly(dT)-albumin. The extended primers only are detected by reaction with a streptavidin-aequorin conjugate. The bound photoprotein aequorin is measured within 3 sec by simply adding Ca2+. We carried out extensive optimization studies of the PEXT reaction and genotyped the six nucleotide variant sites using blood specimens from 27 normal DNA samples. The results of the proposed method agreed entirely with the sequencing data.

Nanoparticle-based DNA biosensor for visual detection of genetically modified organisms.

Although screening of raw ingredients and food products for genetically modified organisms (GMO) may be accomplished by detecting either the exogenous DNA or the novel protein, DNA is the preferred analyte because of its superior stability during food processing. The development of DNA biosensors is of increasing importance due to the growing demand for rapid and reliable methods for GMO detection. We report the first DNA biosensor in a dry-reagent dipstick configuration for visual detection and confirmation of GMO-related sequences by hybridization within minutes. The sensor is disposable and does not require special instrumentation. It detects the 35S promoter and nopaline synthase (NOS) terminator sequences that are present in the majority of transgenic plants. The target sequences are amplified by the polymerase chain reaction (PCR) and hybridized (7min) with probes bearing oligo(dA) tail. The biotinylated product is applied to the sensor followed by immersion in the appropriate buffer. Migration of the buffer rehydrates gold nanoparticles conjugated to oligo(dT), which hybridize with the oligo(dA) tails. The hybrids are captured by immobilized streptavidin at the test zone of the sensor giving a characteristic red line due to the accumulation of the nanoparticles. The excess of nanoparticle conjugates are captured at the control zone by immobilized oligo(dA) strands. Amplified 35S or NOS DNA is detectable at 0.16nM. Soybean powder certified reference material with 0.1% GMO content is clearly detectable after 35 and 40 amplification cycles for 35S and NOS sequence, respectively. The sensor was also applied to real samples from various sources.