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Analytical and Bioanalytical Chemistry Jul 2009In this work, we have studied the effect of different probe lengths and surface densities on the hybridization of a 181-bp polymerase chain reaction product to probes...
In this work, we have studied the effect of different probe lengths and surface densities on the hybridization of a 181-bp polymerase chain reaction product to probes tethered onto magnetic microparticles. Hybridization was shown to be favored by longer probes but only at probe surface densities where probe-to-probe interactions are absent. From these results, a simple rule was inferred for determining maximum surface densities above which hybridization signals decreased. According to this rule, if the average surface area occupied by an immobilized probe (Sigma) is larger than the projected surface area of each tethered probe molecule (S(ss)), hybridization efficiency increases with surface density, whereas the reverse occurs when Sigma-S(ss) < 0.
Topics: DNA; DNA Probes; Magnetics; Nucleic Acid Hybridization; Surface Properties
PubMed: 19462163
DOI: 10.1007/s00216-009-2843-4 -
Molecular and Cellular Probes Dec 1995Strand displacement amplification (SDA) is an isothermal, in vitro method of amplifying a target DNA sequence. We performed SDA in the presence of a...
Strand displacement amplification (SDA) is an isothermal, in vitro method of amplifying a target DNA sequence. We performed SDA in the presence of a 5'-32P-oligodeoxynucleotide detector probe that contains a target binding sequence at its 3'-end and a recognition site for the restriction enzyme HincII at its 5'-end which is not homologous to the target sequence. The single-stranded probe hybridizes to the rising concentration of amplified product during SDA and is converted to a fully double-stranded form that is cleaved by HincII, releasing a 32P-labelled 5-mer fragment. Uncleaved probe (42-mer) and cleaved probe (5-mer) were separated by either gel electrophoresis or size exclusion filtration using a commercially available microcentrifuge device. The combined SDA/filtration protocol is simple and provides detection of as few as 10 molecules of target DNA. We applied the technique to detection of M. tuberculosis DNA.
Topics: Base Sequence; Centrifugation; DNA; DNA Polymerase I; DNA Probes; DNA, Bacterial; DNA, Single-Stranded; Deoxyribonucleases, Type II Site-Specific; Electrophoresis, Polyacrylamide Gel; Microchemistry; Molecular Sequence Data; Mycobacterium tuberculosis; Nucleic Acid Amplification Techniques; Nucleic Acid Denaturation; Phosphorus Radioisotopes; Ultrafiltration
PubMed: 8808310
DOI: 10.1006/mcpr.1995.0062 -
Biosensors & Bioelectronics Sep 2022In situ imaging of DNA repair enzymes in living cells gives important insights to diagnosis and explore the formation of various diseases. Fluorescent probes have become...
In situ imaging of DNA repair enzymes in living cells gives important insights to diagnosis and explore the formation of various diseases. Fluorescent probes have become a powerful and widely used technique for their high sensitivity and real-time capabilities, but empirical design and optimization of the corresponding probes can be blind and time-consuming. Herein, we report a strategy combining experimental studies with molecular simulation techniques for the rapid and rational design of sensitive fluorescent DNA probes for a representative DNA repair enzyme human apurinic/apyrimidinic endonuclease 1 (APE1). Extended-system Adaptive Biasing Force (eABF) was applied to study the interaction mechanism between DNA probes with respect to the enzyme, based on which a novel sensitive DNA probe was designed efficiently and economically. Product inhibition effect which significantly limited the sensitivity of existing probes was eliminated by decreasing the key interactions between DNA probe products and enzyme. Experimental mechanism studies showed the existence of intramolecular hairpin structure in DNA probes is important for the recognition of APE1 and elimination of product inhibition, which is in consistent with the simulations. The obtained fluorescent DNA nanoprobe (Nanoprobe N) showed a high sensitivity for APE1 with the detection limit as low as 0.5 U/L (∼0.018 pM), and the Nanoprobe N could effectively respond to the variation of APE1 within cells and distinguish cancer cells from normal cells. This work not only demonstrated the effectiveness of molecular simulations in probe design, but also provided a reliable platform for accurate imaging of APE1 and effectors screening at single-cell level.
Topics: Biosensing Techniques; Computer Simulation; DNA; DNA Probes; DNA Repair; DNA-(Apurinic or Apyrimidinic Site) Lyase; Humans; Optical Imaging
PubMed: 35609451
DOI: 10.1016/j.bios.2022.114360 -
Analytica Chimica Acta Jun 2021Single nucleotide variants (SNVs) have emerged as increasingly important biomarkers, particularly in the diagnosis and prognosis of cancers. However, most SNVs are...
Single nucleotide variants (SNVs) have emerged as increasingly important biomarkers, particularly in the diagnosis and prognosis of cancers. However, most SNVs are rarely detected in blood samples from cancer patients as they are surrounded by abundant concomitant wild-type nucleic acids. Herein, we design a system that features a combination of competitive DNA probe system (CDPS) and duplex-specific nuclease (DSN) that we referred to as CAD. A theoretical model was established for the CAD system based on reaction networks. Guided by the theoretical model, we found that a minor loss in sensitivity significantly improved the specificity of the system, thus creating a theoretical discrimination factor (DF) > 100 for most conditions. This non-equivalent tradeoff between sensitivity and specificity provides a new concept for the analysis of rare DNA-sequence variants. As a demonstration of practicality, we applied as-proposed CAD system to identify low variant allele frequency (VAF) in a synthetic template (0.1% VAF) and human genomic DNA (1% VAF). This work promises complete guidance for the design of enzyme-based nucleic acid analysis.
Topics: DNA; DNA Probes; Endonucleases; Humans; Nucleic Acids; Nucleotides; Polymorphism, Single Nucleotide
PubMed: 34023002
DOI: 10.1016/j.aca.2021.338545 -
Biosensors & Bioelectronics Feb 2015Highly sensitive detection of transcription factors (TF) is essential to proteome and genomics research as well as clinical diagnosis. We describe herein a novel...
Highly sensitive detection of transcription factors (TF) is essential to proteome and genomics research as well as clinical diagnosis. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, quantitative, and inexpensive detection of TF. The strategy consists of a hairpin DNA probe containing a TF binding sequence for target TF, a dumbbell-shaped probe, a primer DNA probe designed partly complementary to hairpin DNA probe, and a dumbbell probe. In the presence of target TF, the binding of the TF with hairpin DNA probe will prohibit the hybridization of the primer DNA probe with the "stem" and "loop" region of the hairpin DNA probe, then the unhybridized region of the primer DNA will hybridize with dumbbell probe, subsequently promote the ligation reaction and the rolling circle amplification (RCA), finally, the RCA products are quantified via the fluorescent intensity of SYBR Green I (SG). Using TATA-binding protein (TBP) as a model transcription factor, the proposed assay system can specifically detect TBP with a detection limit as low as 40.7 fM, and with a linear range from 100 fM to 1 nM. Moreover, this assay related DNA probe does not involve any modification and the whole assay proceeds in one tube, which makes the assay simple and low cost. It is expected to become a powerful tool for bioanalysis and clinic diagnostic application.
Topics: Biosensing Techniques; DNA Probes; Nucleic Acid Amplification Techniques; Sensitivity and Specificity; Spectrometry, Fluorescence; Transcription Factors
PubMed: 25299987
DOI: 10.1016/j.bios.2014.09.068 -
Analytical Biochemistry Sep 2014HIV-1 integrase is a key enzyme for retroviral replication. The integrase can perform a palindrome cleavage reaction. In this work, a hairpin DNA probe with a...
HIV-1 integrase is a key enzyme for retroviral replication. The integrase can perform a palindrome cleavage reaction. In this work, a hairpin DNA probe with a palindromic DNA sequence mimicking the HIV-1 LTR-LTR junction of the 2-LTR circles in its long stem was designed for fluorescence detection of specific restriction-like cleavage activity of HIV-1 integrase. Results showed that the designed probe could be recognized and cleaved by HIV-1 integrase. The palindrome cleavage reaction can be monitored according to the increase in fluorescent signal. The assay can be applied to real-time detection of palindrome cleavage of HIV-1 integrase with advantages of simplicity, high sensitivity, and specificity.
Topics: Base Sequence; DNA Probes; Enzyme Assays; HIV Integrase; HIV Long Terminal Repeat; HIV-1; Inverted Repeat Sequences; Spectrometry, Fluorescence
PubMed: 24862436
DOI: 10.1016/j.ab.2014.05.010 -
Analytica Chimica Acta Oct 2023We describe a novel lateral flow DNA biosensor (LFDB) based on carbon nanotube (CNT) and triple helix DNA (THD). The carboxylated CNT was first conjugated with...
We describe a novel lateral flow DNA biosensor (LFDB) based on carbon nanotube (CNT) and triple helix DNA (THD). The carboxylated CNT was first conjugated with amine-modified auxiliary single-stranded DNA probe (P) by dehydration reaction and used as signal probe. A main DNA probe (P) was introduced to react with the P and formed the THD on the CNT surface. Because of the large spatial effect, P was in an inactive state and cannot hybridize with the capture DNA probe (P) fixed on the LFDB test area. When the target DNA was present, P in the triple helix DNA hybridized with the target DNA due to the stronger base action, and the decomposition of the triple helix structure exposed P. Therefore, P on CNT surface was activated to hybridize with P. The CNT along with P was thus captured at the test area and accumulated to show a black line, which can be observed by naked eye for qualitative analysis and recorded with a portable grayscale reader for quantitative analysis. Single-stranded DNA was used as a target to prove the feasibility of the model. Under the best experimental conditions, the THD-CNT based LFDB was able to detect the lowest DNA concentration of 15 pM, which is 2.67 times better than that of the traditional duplex CNT-based LFDB. It should be noted that the LFDB based on THD functionalized CNT can differentiate between one-base-mismatched DNA and the complementary target DNA, can detected target DNA in 10% human serum, and can be employed as a versatile platform to detect various target (proteins, small molecular) by changing the sequence of P. This biosensor platform has enormous potential in the point-of-care detection of a rich diversity of analytes for clinical diagnosis and biomedical research.
Topics: Humans; Nucleic Acids; Nanotubes, Carbon; DNA, Single-Stranded; DNA; DNA Probes; Biosensing Techniques; DNA, Complementary
PubMed: 37573103
DOI: 10.1016/j.aca.2023.341604 -
Analytical Chemistry Feb 2022Nuclease-resistant assay probes are of significant importance for biochemical analysis and disease diagnosis. In this contribution, a reconfigurable lipidic...
Nuclease-resistant assay probes are of significant importance for biochemical analysis and disease diagnosis. In this contribution, a reconfigurable lipidic moiety-attached DNA nanoparticle (LDN) is constructed from a cholesterol-conjugated multifunctional hairpin-type DNA probe (Chol-DP) by hydrophobicity-mediated self-assembly. The LDN holds high serum stability and displays a low false-positive signal even in a complex biological milieu. The hydrophobic cholesterol moiety enables the hydrophobicity-mediated assembly, while hydrophilic DNA sequence serves as a recognition element and a polymerization template. The initiator-activated strand displacement amplification (SDA) reaction can convert the hairpin-shaped probe into rigid double-stranded DNA (dsDNA), causing the conformational rearrangement-based LDN swelling that can be used to reliably and fluorescently signal the cancer-related p53 gene. The size increase and structural reconfiguration are confirmed by dynamic light scattering (DLS) analysis and confocal microscopy imaging, respectively. Target p53 is specifically detected down to 10 pM. The whole assay process involved only several simple mixing steps. Recovery test and blind test further confirm the feasibility of the use of the LDN for the detection of target DNA in a complex biological milieu, indicating a promising nanotool for biomedical applications.
Topics: Base Sequence; Biosensing Techniques; DNA; DNA Probes; Humans; Nanoparticles; Neoplasms; Nucleic Acid Amplification Techniques
PubMed: 35099191
DOI: 10.1021/acs.analchem.1c03598 -
Biosensors & Bioelectronics Jan 2014A facile strategy for the assay of target miRNA using fluorescent silver nanoclusters (AgNCs) has been described. Due to the preferable interaction between cytosine...
A facile strategy for the assay of target miRNA using fluorescent silver nanoclusters (AgNCs) has been described. Due to the preferable interaction between cytosine residues and Ag(+), a short cytosine-rich oligonucleotide (ODN) with only six bases 5'-TCCCCC-3' served as an efficient scaffold for the creation of the AgNCs. The AgNCs displayed a bright red emission when excited at 545nm. Such ODN base-stabilized AgNCs have been exploited for miRNA sensing. Overhangs of TCC at the 5' end (5'-TCC) and CCC at the 3' end (CCC-3') (denoted as 5'-TCC/CCC-3') appended to the hairpin ODN probe which also contains recognition sequences for target miRNA were included. Interestingly, the AgNCs/hairpin ODN probe showed similar spectral properties as that templated by 5'-TCCCCC-3'. The formation of the hairpin ODN probe/miRNA duplex separated the 5'-TCC/CCC-3' overhangs, thus disturbing the optical property or structure of the AgNCs. As a result, fluorescence quenching of the AgNCs/hairpin ODN probe was obtained, which allows for facile determination of target miRNA. The proposed method is simple and cost-effective, holding great promise for clinical applications.
Topics: Base Sequence; Biosensing Techniques; Cytosine; DNA Probes; MicroRNAs; Nanostructures; Oligonucleotide Probes; Sensitivity and Specificity; Silver; Spectrometry, Fluorescence
PubMed: 23932977
DOI: 10.1016/j.bios.2013.07.036 -
Nucleic Acids Research 2007Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually...
Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.
Topics: Base Pair Mismatch; Base Sequence; DNA Probes; Fluorescent Dyes; Genetic Variation; HIV-1; Reverse Transcriptase Polymerase Chain Reaction; Thermodynamics
PubMed: 17693434
DOI: 10.1093/nar/gkm551