Single nucleotide recognition using a probes-on-carrier DNA chip (original) (raw)
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DNA-based biosensor platforms for the detection of TP53 mutation
Sensors and Actuators B: Chemical, 2012
A DNA-based assay for the detection of one-point mutation in TP53 gene, responsible for lung cancer, was developed using a surface plasmon resonance (SPR) and a quartz crystal microbalance (QCM) biosensor systems. Amine coupling was employed for the immobilization of NeutrAvidin on thiol-derivatised surface to capture the biotinylated target sequence. Two targets sequences and one control DNA sequence were investigated including, a fully complementary (30 mer), onepoint mutation and a non-complimentary DNA using hybridization with a detection probe strand (27 mer). The most appropriate surface coating was also examined for both sensor platforms with hybridization and single nucleotide polymorphism (SNP) detection efficiency were then compare. A 0.03-2 µM concentration range of detection probe was detected using the SPR and QCM sensors on wild and mutant type target surfaces. The linear regression analysis was performed for both sensors resulting in a R 2 value for the SPR assay of 0.985 and 0.993 for perfect and mismatch reaction and of 0.978 and 0.976 for the QCM assay, respectively. The obtained results demonstrate that the used approach represents a very promising future method for the detection of one-point mutation in genetic-based health problem with highly sensitive, specific, and real-time analysis.
A novel optical biosensor format for the detection of clinically relevant TP53 mutations
Biosensors & Bioelectronics, 2005
The TP53 gene has been the subject of intense research since the realisation that inactivation of this gene is common to most cancer types. Numerous publications have linked TP53 mutations in general or at specific locations to patient prognosis and therapy response. The findings of many studies using general approaches such as immunohistochemistry or sequencing are contradictory. However the detection of specific mutations, especially those occurring in the structurally important L2 and L3 zinc binding domains, which are the most common sites of TP53 mutations, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. In this study, the TI-SPR-1 surface plasmon resonance system and Texas Instruments Spreeta TM chips were used to develop a DNA biosensor based on thiolated probes complementary to these domains. The sensors were able to detect these mutations in both oligonucleotides and PCR products with normal and mutant TP53 DNA, but the difference in hybridisation signal was small. Preliminary experiments to enhance the signal using Escherichia coli mismatch repair proteins, MutS and single strand binding protein were carried out. It was found that MutS was unable to bind to mismatch oligonucleotides, but single strand binding protein was able to bind to single stranded probes which had not hybridised to the target, resulting in a 3-fold increase in the sensitivity of the biosensor. While further work needs to be carried out to optimise the system, these preliminary experiments indicate that the TI-SPR-1 can be used for the detection of clinically relevant mutations in the TP53 gene and that the sensitivity can be increased significantly using single strand binding protein. This system has a number of advantages over current mutation detection technologies, including lower cost, ease of sensor preparation and measurement procedures, technical simplicity and increased speed due to the lack of need for gel electrophoresis.
ELECTROPHORESIS, 2003
We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.
On-chip detection of a single nucleotide polymorphism without polymerase amplification
Nano Research, 2014
A nanoparticle-assembled photonic crystal (PC) array was used to detect single nucleotide polymorphism (SNP). The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair (bp) difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease (PKD) with a sensitivity of 0.9 fg/mL (50 aM)-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA (gDNA) extracted from the urine and blood of two PKD-wild type and three PKD positive cats. The standard curves for PKD positive (PKD + ) and negative (PKD -) DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection (LOD) was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction (PCR). The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analysis is desirable.
Analytical and Bioanalytical Chemistry, 2004
Single nucleotide polymorphism (SNP) analysis at the point of care requires a low cost detection technology that is capable of miniaturization, multiplexing, and high sensitivity. Direct current electrical detection (DCED) of DNA following nanoparticle labeling and silver enhancement is a promising candidate technology for point-of-care diagnostics. In this work we present, for the first time, SNP analysis in PCR products from patient samples using DCED, taking this platform technology a step closer to practical application. We developed a silane functionalized polymer for coating of biochip surfaces. This polymeric coating is stable under harsh conditions and has exceptionally high binding capacity. Allele-specific oligonucleotide probes were immobilized on chips coated with this polymer. Biotinylated PCR products of the human cholesteryl ester transfer protein gene from different patients were hybridized to the chips, labeled with gold nanoparticles, and autometallographically enhanced. The chips were scanned for DC electrical resistance by applying movable electrodes to the surface. Eighteen of nineteen patient samples were assigned the correct genotype. Our results demonstrate that SNP analysis of patient samples is feasible with DCED.
Blinded Study Microchip Electrophoresis Of P53 Gene
Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (497%) in order to prevent misdiagnoses. Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNAbased, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)-SSCP/HA. We demonstrate the ability of ME-SSCP/HA to detect with 98% sensitivity and specificity 4100 samples from the p53 gene exons 5-9 in a blinded study in an analysis time of o10 min.
APEX protocol implementation on a lab-on-a-chip for SNPs detection
Microelectronic Engineering, 2008
In this work a detection module for the single nucleotide polymorphisms (SNPs) detection was realised. In particular arrayed primer extension (APEX) was selected as innovative method for SNPs detection and this protocol was scaled down following a micro total analysis approach in order to fabricate a lab-on-a-chip (LOC). Finite element analysis and behavioural simulations with commercial tools to properly design the microfluidic circuitry have preceded the technological processes for the production of the device. The fluidic was designed to contain 5 ll of DNA and reagents that are inserted through three inlets. The layout includes two mixers and a sealed reaction chamber. Glass/silicon prototypes were fabricated with the employment of micro-electro-mechanical-system (MEMS) processes. The devices were tested with APEX biological protocols customized for the scaled volumes. Furthermore, in order to demonstrate the absence of any negative interaction between the chip and the APEX reagents, different chips were tested in intermediate steps together with the protocol executed in standard conditions. The final result demonstrates that the thermo sequenase extended the probes with the dideoxynucleotides modified with Cy5 fluorophore, thus indicating the possibility of implementation of the APEX protocol on LOC devices.
Applications of DNA chips for genomic analysis
Molecular Psychiatry, 1998
A major frontier in medical genetics is the definition of the molecular basis of multifactorial diseases. This is especially relevant in the field of clinical psychiatry where the majority of common disorders display complex inheritance patterns, and are further influenced by environmental interactions. New technologies are needed to help address the pressing needs for discovering and deciphering the nature of such disease-associated genes. One such technology which has emerged within the past 3 years involves hybridization-based nucleic acid array (DNA chip) analysis. This technology has the potential to have a lasting impact on diverse genomic-based applications such as large-scale gene mapping studies, mutational analysis, and global expression level monitoring of all human genes. In this review we will describe the fundamental principles behind nucleic acid array-based assays, while focusing on their applications towards genome-wide DNA and RNA analysis. The current capabilities and limitations of these technologies will be discussed, with a focus on areas where future development will be needed for DNA chip-based assays to achieve their full potential.