Linear DNA Probes Research Articles

Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR

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.

Partially double-stranded linear DNA probes: Novel design for sensitive detection of genetically polymorphic targets

Genetically polymorphic targets present a significant challenge to the reliability of detection and quantification by nucleic acid-based assays. A probe system with enhanced mismatch tolerance would be advantageous for such applications. The present study introduces a novel class of DNA probes, designated as partially double-stranded linear probes, composed of a long target-specific strand 5′ labeled with a fluorophore and a markedly shorter quencher strand, complementary to the 5′ end of the target-specific strand, that is 3′ end-labeled with a quencher moiety. The utility of this probe system for sensitive detection of amplification products was demonstrated in a real-time PCR format. Comparison of multiple partially double-stranded linear probe combinations revealed that increased asymmetry in strand length was associated with improved mismatch tolerance. Notably, for a 45-mer/11-mer combination, the difference in threshold cycle values obtained for a perfectly matched target and one containing six mismatches was <1.5 cycles. The capacity for superior mismatch tolerance, ease of design, simplicity and flexibility of application are characteristics that make this new class of probes a desirable alternative for homogeneous detection of targets with a high level of genetic heterogeneity.

Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry

Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry.

Breast Cancer–Specific mRNA Transcripts Presence in Peripheral Blood After Adjuvant Chemotherapy Predicts Poor Survival Among High-Risk Breast Cancer Patients Treated With High-Dose Chemotherapy With Peripheral Blood Stem Cell Support

To study the prognostic significance of the presence of breast cancer-specific mRNA transcripts in peripheral blood (PB), defined by serial analysis of gene expression, in high-risk breast cancer (HRBC) patients undergoing high-dose chemotherapy after receiving adjuvant chemotherapy. From 1994 to 2000, 84 HRBC patients (median age, 44 years; > 10 nodes; 74%) received adjuvant chemotherapy (fluorouracil, epirubicin, and cyclophosphamide for six cycles [83%] or doxorubicin and cyclophosphamide followed by paclitaxel) before undergoing one course of cyclophosphamide plus thiotepa plus carboplatin (STAMP V). Radiotherapy or hormone therapy was administered whenever indicated. Aliquots of apheresis-mononuclear blood cells were frozen from each patient. mRNA was isolated using an automatic nucleic acid extractor based on the magnetic beads technology; reverse transcription was performed using random hexamers. Cytokeratin 19, HER-2, P1B, PS2, and EGP2 transcripts were quantified to B-glucuronidase by real-time polymerase chain reaction (RT-PCR) using a linear DNA probe marked with a quencher and reporter fluorophores used in RT-PCR. Presence of PB micrometastases, estrogen receptor and progesterone receptor status, tumor size, age, tumor grade, number of nodes affected, and treatment with paclitaxel were included in the statistical analysis. Median follow-up was 68.3 months (range, 6 months to 103 months). Forty-seven relapses (56%) and 35 deaths (41.7%) were registered. Both tumor size and presence of micrometastases reached statistical significance according to the Cox multivariate model. Relapse hazard ratio (HR) for those patients with PB micrometastases was 269% (P = .006); death HR, 300% (P = .011). Time relapse was 53 months longer for patients without micrometastases: 31.3 v 84.2 months (P = .021). PB micrometastases presence after adjuvant chemotherapy predicts both relapse and death more powerful than classical factors in HRBC patients undergoing high-dose chemotherapy. Micrometastases search using a gene panel appears to be a more accurate procedure than classical approaches involving only one or two genes.

An efficient method for recovery of target ssDNA based on amino-modified silica-coated magnetic nanoparticles

In this paper, an improved recovery method for target ssDNA using amino-modified silica-coated magnetic nanoparticles (ASMNPs) is reported. This method takes advantages of the amino-modified silica-coated magnetic nanoparticles prepared using water-in-oil microemulsion technique, which employs amino-modified silica as the shell and iron oxide as the core of the magnetic nanoparticles. The nanoparticles have a silica surface with amino groups and can be conjugated with any desired bio-molecules through many existing amino group chemistry. In this research, a linear DNA probe was immobilized onto nanoparticles through streptavidin conjugation using covalent bonds. A target ssDNA(I) (5′-TMR-CGCATAGGGCCTCGTGATAC-3′) has been successfully recovered from a crude sample under a magnet field through their special recognition and hybridization. A designed ssDNA fragment of severe acute respiratory syndrome (SARS) virus at a much lower concentration than the target ssDNA(I) was also recovered with high efficiency and good selectivity.

Electrochemical detection of the immobilization and hybridization of unlabeled linear and hairpin DNA on gold

Unlabeled hairpin and linear DNA probes have been immobilized on polycrystalline gold electrodes by forming mixed SAMs of thiolated single-stranded oligonucleotides (HS-ssDNA) and a spacer thiol (4-mercaptobutan-1-ol) by spontaneous coadsorption. Chronocoulometry was used to determine the amount of DNA present in the mixed SAM. It was found that linear ssDNA achieves a higher surface concentration compared to hairpin ssDNA under the same deposition conditions. The hybridization behaviour of these SAMs was analysed by impedance spectroscopy in the presence of [Fe(CN) 6] 3−/4−. Addition of the complementary oligonucleotide to the linear ssDNA modified gold electrode resulted in an increase of the electron transfer resistance R ct, while it decreased in the case of the hairpin DNA probe. These results are due to the smaller surface coverage and the conformational change of the DNA hairpin probe upon hybridization. Hybridization experiments with a non-complementary sequence resulted in no change of the impedance spectra.

Monitoring molecular beacon/DNA interactions using atomic force microscopy.

The molecular beacon (MB) is a new fluorescence probe containing a single-stranded oligonucleotide with a probe sequence embedded in complementary sequences that form a hairpin stem. Due to the inherent fluorescent signal transduction mechanism, an MB functions as a sensitive probe with a high signal-to-background ratio for real-time monitoring and provides a variety of exciting opportunities in DNA, RNA, and protein studies. To better understand the properties of MBs, the specific interactions between MB and target DNA (complementary and one-base mismatch) have been directly investigated by atomic force microscopy. The interaction force between a linear DNA probe and the target DNA was also detected and compared to that between MB and target DNA. The results demonstrate the high specificity of the MB/target DNA compared to the linear DNA/target DNA interaction.

Detection of activity of telomerase in tumor cells using fiber optical biosensors

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80–90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 °C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.

Rapid DNA fiber technique for size measurements of linear and circular DNA probes.

Rapid DNA fiber technique for size measurements of linear and circular DNA probes.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes.

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T(2)-biotin.dT-T(2) loop. The third base was a biotinylated uracil (U(B)) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3' dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3' end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5'-FITC, or radiolabeled with [gamma-(33)P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45 degrees C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin-target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

A new label technology for the detection of specific polymerase chain reaction products in a closed tube.

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5'-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5'-->3' exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10-10(7)initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5'-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.

RecA-mediated affinity capture: a method for full-length cDNA cloning.

We describe an improved method for rapid cloning of full-length cDNA from cDNA libraries. This approach is based on the ability of Escherichia coli RecA protein to form a stable nucleoprotein complex with a linear single-stranded DNA probe and homologous sequences in circular double-stranded DNA. Hybridization of RecA-coated biotinylated DNA probes to homologous plasmid DNA creates triple-stranded complexes, which are then captured on streptavidin-coated magnetic beads. Following magnetic separation of the hybrid molecules, the enriched plasmid population is recovered by alkaline treatment, precipitated, resuspended and used to transform bacteria. Typically, many clones can then be recovered by colony hybridization screening of a single plate of the enriched library. We have used this technology to clone full-length and alternatively spliced forms of the human bcl-xL cDNA from a human liver cDNA library.

Differential effects of heavy metals on the binding of Xenopus upstream binding factor( xUBF) to DNA

Xenopus upstream binding factor ( xUBF) is a transcription factor for RNA polymerase I which contains multiple DNA-binding motifs. Among these DNA-binding motifs,HMG box I is essential for promoting RNA polymerase I-dependent rRNA gene transcription. Gel shift assay indicated that the binding of recombinant HMG box I to a 136-bp linear DNA probe was significantly inhibited by Cd 2+ at 1 μM.The formation of larger protein-DNA complexes was particularly sensitive to Cd 2+.The intereaction between HMG box I and DNA was completely inhibited by 10 μM of Cd 2+,yet this intereaction was not inhibited by the same concentration of Ca 2+. Hg 2+ at 0.1μM began to cause abnormal band shifting, and protein-DNA bands disappeared to the wells of a polyacrylamide gel in the presence of 10 μM of Hg 2+, reflecting that a drastic change in the conformation of HMG box I-DNA had occurred. The binding of HMG box I to DNA was slightly disturbed by As 3+ at 1 μM and was significantly affected at 10 μM.Our results suggest that inhibition of the normal binding of UBF to its target DNA may be one of the mechanisms of heavy metal-induced inhbbtion of RNA synthesis.

Evidence for dna endonuclease activity in nuclear extracts from mosquito cells

We describe a deoxyribonuclease activity from nuclear protein extracts of cultured Aedes albopictus mosquito cells. The nuclease cleaved linear and circular double-stranded DNA, first generating 3′ OH single-stranded nicks followed by second strand cleavage, but had little or no exonucleolytic activity. Detection of this activity was optimal at pH 7.1, in the presence of a divalent cation (Mg 2+, Ca 2+, Mn 2+, Ba 2+). In the presence of Mg 2+, Zn 2+, Hg 2+ and Cu 2+ inhibited activity, sulfhydryl reagents and ATP had no effect. At physiological temperatures (18–35°C), linear double-stranded DNA probes were preferentially cleaved near sites containing 3-6 consecutive deoxyadenine/thymine base pairs. Results from salt dependency and drug inhibition studies, combined with inspection of DNA sequence, suggested that DNA structure is among the parameters that determine preferred cleavage sites.

Rapid plasmid library screening using RecA-coated biotinylated probes.

A method for the rapid physical isolation of recombinant plasmids of interest from a mixture of plasmids such as a plasmid cDNA library is presented. This method utilizes the ability of RecA protein to form stable complexes between linear single-stranded and circular double-stranded DNA molecules sharing sequence homology, and procedures allowing isolation of biotinylated nucleic acid. Biotinylated linear DNA probes coated with RecA have been used to screen reconstituted plasmid libraries consisting of two plasmid species, one homologous and the other heterologous to the probe. When the link between biotin and the nucleotide base could be cleaved by reducing agents, the complex was purified by streptavidin-agarose chromatography and the recovered plasmid was propagated in Escherichia coli. When the link was not cleavable the complex was bound to avidin in solution and purified by cupric iminodiacetic acid-agarose chromatography. The complex was then dissociated and the plasmids were propagated in E. coli. With either protocol, homologous plasmid recovery was between 10% and 20%, and enrichment was between 10(4)- and 10(5)-fold. Potential applications and extensions of this method, such as plasmid, cosmid, and phage library screening and facilitation of physical mapping of macroregions of mammalian genomes are presented and discussed.