Increasing the analytical sensitivity by oligonucleotides modified with para- and ortho-twisted intercalating nucleic acids--TINA

The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5' and 3' termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch--in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.     

Photo-cross-linkable oligonucleotide probes for in situ hybridization assays

In situ hybridization techniques have been an important research tool since first introduced 30 years ago, and more recently clinical applications have been expanding greatly. Still, further improvements in the assay sensitivity and protocols that are amenable to routine clinical use are desired. We use a novel photo-cross-linking technology to irreversibly bind short oligonucleotide probes to the target sequence following a hybridization period. The cross-linking agent is incorporated into the backbone of the probe and is activated to react with pyrimidines in the opposite strand by near-UV (300-370 nm) irradiation. By locking the probe to the target, very stringent wash conditions can be used that would otherwise completely remove probes that are hybridized but not cross-linked to the target. Consequently, the probe-specific signal is maximized, while the background signal is minimized to the greatest extent possible with the stringency of the wash. The use of short, photo-cross-linkable probes presents a new strategy for maximizing the sensitivity of probe hybridization or signal amplification-based in situ techniques.     

DNA-mounted self-assembly: new approaches for genomic analysis and SNP detection

This article presents an overview of new emerging approaches for nucleic acid detection via hybridization techniques that can potentially be applied to genomic analysis and SNP identification in clinical diagnostics. Despite the availability of a diverse variety of SNP genotyping technologies on the diagnostic market, none has truly succeeded in dominating its competitors thus far. Having been designed for specific diagnostic purposes or clinical applications, each of the existing bio-assay systems (briefly outlined here) is usually limited to a relatively narrow aspect or format of nucleic acid detection, and thus cannot entirely satisfy all the varieties of commercial requirements and clinical demands. This drives the diagnostic sector to pursue novel, cost-effective approaches to ensure rapid and reliable identification of pathogenic or hereditary human diseases. Hence, the purpose of this review is to highlight some new strategic directions in DNA detection technologies in order to inspire development of novel molecular diagnostic tools and bio-assay systems with superior reliability, reproducibility, robustness, accuracy and sensitivity at lower assay cost. One approach to improving the sensitivity of an assay to confidently discriminate between single point mutations is based on the use of target assembled, split-probe systems, which constitutes the main focus of this review.     

Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.     

High-sensitivity hybridization assay for quantitation of residual E. coli DNA

Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's genomic DNA have traditionally been used for products derivedfrom bacterial expression systems to obtain the required low picogram sensitivity. Nonradioactive methods, while desirable to eliminate radioactive waste disposal and safety issues, typically suffer from poor sensitivity and high backgrounds. We report the development of a suitably sensitive, nonradioactive assay to quantitate residual E. coli DNA levels in purified protein drugs by means of a slot-blot hybridization method. The assay utilizes digoxigenin-labeled E. coli DNA probes and SuperSignal chemiluminescent substrate. The optimized chemiluminescent hybridization assay has both low background and high sensitivity, allowing routine detection of 2.5 pg E. coli DNA. The method can be tailored for detection/quantitation of DNA contamination in recombinant protein products expressed in E. coli or other bacterial expression systems.     

Clinical application of array-based comparative genomic hybridization for the identification of prognostically important genetic alterations in chronic lymphocytic leukemia

Genomic aberrations have increasingly gained attention as prognostic markers in B-cell chronic lymphocytic leukemia (CLL). Fluorescence in situ hybridization (FISH) has improved the detection rate of genomic alterations in CLL from approximately 50% using conventional cytogenetics to greater than 80%. More recently, array comparative genomic hybridization (CGH) has gained popularity as a clinical tool that can be applied to detect genomic gains and losses of prognostic importance in CLL. Array CGH and FISH are particularly useful in CLL because genomic gains and losses are key events with both biologic and prognostic significance, while balanced translocations have limited prognostic value. Although FISH has a higher technical sensitivity, it requires separate, targeted hybridizations for the detection of alterations at genomic loci of interest. Array CGH, on the other hand, has the ability to provide a genome-wide survey of genomic aberrations with a single hybridization reaction. Array CGH is expanding the known genomic regions of importance in CLL and allows these regions to be evaluated in the context of a genome-wide perspective. Ongoing clinical trials are evaluating the use of genomic aberrations as tools for risk-stratifying patients for therapy, thus increasing the need for reliable and high-yield methods to detect these genomic changes. In this review, we consider the use of array CGH as a clinical tool for the identification of genomic alterations with prognostic significance in CLL, and suggest ways to integrate this test into the clinical molecular diagnostic laboratory work flow.     

Computer simulation study of molecular recognition in model DNA microarrays

DNA microarrays have been widely adopted by the scientific community for a variety of applications. To improve the performance of microarrays there is a need for a fundamental understanding of the interplay between the various factors that affect microarray sensitivity and specificity. We use lattice Monte Carlo simulations to study the thermodynamics and kinetics of hybridization of single-stranded target genes in solution with complementary probe DNA molecules immobilized on a microarray surface. The target molecules in our system contain 48 segments and the probes tethered on a hard surface contain 8-24 segments. The segments on the probe and target are distinct and each segment represents a sequence of nucleotides ( approximately 11 nucleotides). Each probe segment interacts exclusively with its unique complementary target segment with a single hybridization energy; all other interactions are zero. We examine how the probe length, temperature, or hybridization energy, and the stretch along the target that the probe segments complement, affect the extent of hybridization. For systems containing single probe and single target molecules, we observe that as the probe length increases, the probability of binding all probe segments to the target decreases, implying that the specificity decreases. We observe that probes 12-16 segments ( approximately 132-176 nucleotides) long gave the highest specificity and sensitivity. This agrees with the experimental results obtained by another research group, who found an optimal probe length of 150 nucleotides. As the hybridization energy increases, the longer probes are able to bind all their segments to the target, thus improving their specificity. The hybridization kinetics reveals that the segments at the ends of the probe are most likely to start the hybridization. The segments toward the center of the probe remain bound to the target for a longer time than the segments at the ends of the probe.     

Integrative genomic analysis of small-cell lung carcinoma reveals correlates of sensitivity to bcl-2 antagonists and uncovers novel chromosomal gains

Cancer is a highly heterogeneous disease in terms of the genetic profile and the response to therapeutics. An early identification of a genomic marker in drug discovery may help select patients that would respond to treatment in clinical trials. Here we suggest coupling compound screening with comparative genomic hybridization analysis of the model systems for early discovery of genomic biomarkers. A Bcl-2 antagonist, ABT-737, has recently been discovered and shown to induce regression of solid tumors, but its activity is limited to a fraction of small-cell lung carcinoma (SCLC) models tested. We used comparative genomic hybridization on high-density single-nucleotide polymorphism genotyping arrays to carry out a genome-wide analysis of 23 SCLC cell lines sensitive and resistant to ABT-737. The screen revealed a number of novel recurrent gene copy number abnormalities, which were also found in an independent data set of 19 SCLC tumors and confirmed by real-time quantitative PCR. A previously unknown amplification was identified on 18q and associated with the sensitivity of SCLC cell lines to ABT-737 and another Bcl-2 antagonist. The region of gain contains Bcl-2 and NOXA, two apoptosis-related genes. Expression microarray profiling showed that the genes residing in the amplified region of 18q are also overexpressed in the sensitive lines relative to the resistant lines. Fluorescence in situ hybridization analysis of tumors revealed that Bcl-2 gain is a frequent event in SCLC. Our findings suggest that 18q21-23 copy number will be a clinically relevant predictor for sensitivity of SCLC to Bcl-2 family inhibitors. The 18q21-23 genomic marker may have a broader application in cancer because Bcl-2 is associated with apoptosis evasion and chemoresistance.     

High-resolution array CGH increases heterogeneity tolerance in the analysis of clinical samples

Recent advances in array comparative genomic hybridization (array CGH) technology are revolutionizing our understanding of tumor genomes. Marker-based arrays enable rapid survey at megabase intervals, while tiling path arrays examine the entire genome in unprecedented detail. Tumor biopsies are typically small and contain infiltrating stromal cells, requiring tedious microdissection. Tissue heterogeneity is a major barrier to high-throughput profiling of tumor genomes and is also an important consideration for the introduction of array CGH to clinical settings. We propose that increasing array resolution will enhance detection sensitivity in mixed tissues and as a result significantly reduce microdissection requirements. In this study, we first simulated normal cell contamination to determine the heterogeneity tolerance of array CGH and then validated this detection sensitivity model on cancer specimens using the newly developed submegabase resolution tiling-set (SMRT) array, which spans the human genome with 32,433 overlapping BAC clones.     

DNA hybridization detection with organic thin film transistors: toward fast and disposable DNA microarray chips

We demonstrate a novel DNA hybridization detection method with organic thin film transistors. DNA molecules are immobilized directly on the surface of organic semiconductors, producing an unambiguous doping-induced threshold voltage shift upon hybridization. With these shifts, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) are differentiated successfully. This method is expected to result in higher sensitivity than the main competitive technology, ISFET-based sensors because of the direct exposure of DNA molecules to sensitive layers. Factors that influence sensor sensitivity have been analyzed and optimum conditions have been determined using statistically designed experiments. Under the optimum conditions, the maximum difference between saturation current ratios caused by ssDNA and dsDNA reaches as high as 70%. In order to make DNA detection fast, we also demonstrate rapid on-chip electrically enhanced hybridization using the TFTs. These technologies together will enable the realization of disposable, rapid-turnaround tools for field-deployable genomic diagnosis.     

Proximal 19q trisomy: a new syndrome of morbid obesity and mental retardation

AIMS: To report on the clinical and metabolic characteristic and the unique chromosomal defect of a mentally retarded and morbidly obese patient. METHODS: A 13-year follow-up, including insulin sensitivity, lipid profile and polysomnography studies and various therapeutic interventions are described. The presence of a supernumerary marker in karyotype preparation was further studied by fluorescence in situ hybridization (FISH). Comparative genomic hybridization (CGH) was used to identify the source of the chromosomal marker. RESULTS: Insulin resistance was found by the homeostatic model assessment (HOMA) and the quantitative insulin sensitivity check index (QUICKI). M-FISH identified euchromatin derived from chromosome 19, and CGH confirmed the FISH results and demonstrated that the supernumerary marker derived from 19q12 to 19q13.2. CONCLUSION: The clinical and metabolic characteristics in association with partial chromosomal trisomy differ our patient from the currently known syndromes of obesity and mental retardation. The metabolic impairments in this case can derive from unbalanced expression of several genes in the 19q12-19q13.2 region, genes that are related to adipose tissue homeostasis and insulin resistance. The clinical and genetic similarities to a previously reported case may suggest that partial 19q trisomy is a new syndrome of obesity and mental retardation.     

Signal amplification at the ultrastructural level using biotinylated tyramides and immunogold detection

 The tyramide amplification technique has recently been developed for signal enhancement in enzyme-linked immunosorbent assays and western blots. This method relies on using labelled tyramides as substrates for peroxidase, resulting in an immobilization of the labelled tyramide residues (tyramide reaction). We succeeded in establishing reliable protocols for the use of the tyramide reaction at the electron microscopic (EM) level. As model systems we chose the visualization of DNA in late spermatocytes, of actin in skeletal muscle, and the visualization of an rDNA probe after DNA-DNA in situ hybridization. We observed a significant increase in signal density after performing the tyramide reaction at the EM level. The tyramide amplification technique at the ultrastructural level therefore appears to be a useful tool to detect even a few epitopes present at the surface of a section as shown after in situ hybridization. It offers advantages over other amplification systems, such as the peroxidase-mediated deposition of diaminobenzidine, because of an increased spatial resolution, whereas specificity and sensitivity are comparable to the conventional immunogold detection method. Accepted: 10 June 1997     

Realities of diagnosing Helicobacter pylori infection in clinical practice: a case for non-invasive indirect methodologies.

BACKGROUND: The current, arbitrarily defined gold standard for the diagnosis of H. pylori infection requires histologic examination of two specially stained antral biopsy specimens. However, routine histology is potentially limited in general clinical practice by both sampling and observer error. The current study was designed to examine the diagnostic performance of invasive and non-invasive H. pylori detection methods that would likely be available in general clinical practice. METHODS: The diagnostic performance of rotating clinical pathology faculty using thiazine staining was compared with that of an expert gastrointestinal pathologist in 38 patients. In situ hybridization stains of adjacent biopsy cuts were also examined by the expert pathologist for further comparison. Receiver operator characteristic (ROC) analysis was performed to evaluate whether the diagnostic performance of the expert pathologist differed depending upon the histologic method employed. A similar analysis was made to evaluate the diagnostic performance of pathology trainees relative to the expert. In the absence of an established invasive gold standard, non-invasive testing methods (rapid serum antibodies, formal Elisa antibodies and carbon-14 urea breath testing) were evaluated in 74 patients by comparison with a gold standard defined using a combination of diagnostic tests. RESULTS: Using either rapid urease testing of biopsy specimens or urea breath testing as the gold standard for comparison, the diagnostic performance of the rotating clinical pathology faculty was inferior to that of the expert gastrointestinal pathologist especially with regard to specificity (e.g., 69 percent for the former versus 88 percent, with the latter relative to rapid urease testing). Although interpretation of in situ hybridization staining by the expert appeared to have an even higher specificity, ROC analysis failed to show a difference. The mean ROC areas for thiazine and in situ hybridization staining for trainee pathologists relative to the expert were 0.88 and 0.94, respectively. In untreated patients, urea breath testing had a sensitivity and specificity of 100 percent as compared with thiazine staining with a sensitivity of 83 percent and a specificity of 97 percent. Post-therapy, breath testing had a sensitivity of 100 percent but a specificity of only 86 percent as compared with invasive testing with a sensitivity and specificity of 100 percent. Rapid serum antibody testing and formal Elisa antibody testing agreed in 93 percent of cases (Kappa 0.78) with the rapid test being correct in three of the four disagreements. CONCLUSIONS: The current study illustrates a number of realities regarding H. pylori diagnosis. There is no diagnostic gold standard in general clinical practice. Accurate interpretation of specially stained slides is a learned activity with a tendency towards overdiagnosis early on. Urea breath testing is likely to be the diagnostic method of choice for untreated patients in general clinical practice although antibody testing is almost as accurate. Rapid antibody tests are at least as accurate as formal Elisa antibody tests. Urea breath testing is useful for confirming cure after therapy, but false-positive results may occur in some patients.     

Matching base-pair number dependence of the kinetics of DNA-DNA hybridization studied by surface plasmon fluorescence spectroscopy

Two single-stranded DNA oligonucleotides consisting of complementary base-pairs can form double strands. This phenomenon is well studied in solutions, however, in order to clarify the physical mechanism of the hybridization occurring at a solid/solution interface, we studied the kinetics by surface plasmon fluorescence spectroscopy (SPFS): one single-stranded oligo-DNA (probe-DNA) was immobilized on the substrate, the other one (target-DNA) labelled with a fluorescent probe was added to the flow cell. After hybridization, the chromophores could be excited by the surface plasmon mode and their fluorescence detected with high sensitivity. The dependence of the k(on) and k(off) rate constants on the length of the hybridizing oligonucleotides was investigated by using a MM0 series (no mismatch) and the kinetics was found to be well described by a Langmuir adsorption model. From these measurements we found that also in the case of surface hybridization the affinity of the duplexes decreases as the number of matching base-pairs decreases from 15 to 10. In order to show that SPFS is the powerful technique with high sensitivity, the hybridization process for mixed target-oligos was measured by SPFS and analyzed by an expanded Langmuir model in which two components of target-oligo can bind to probe-DNA at the sensor surface competitively. Two sets of the k(on) and k(off) obtained from the experiment are successfully consistent with the k(on) and k(off) obtained from experiments for single (pure) target-DNA.     

Comparison of surface plasmon resonance spectroscopy and quartz crystal microbalance techniques for studying DNA assembly and hybridization

In this study we evaluate the strengths and weaknesses of surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) technique for studying DNA assembly and hybridization reactions. Specifically, we apply in parallel an SPR instrument and a 5 MHz QCM device with dissipation monitoring (QCM-D) to monitor the assembly of biotinylated DNA (biotin-DNA) on a streptavidin-modified surface and the subsequent target DNA hybridization. Through the parallel measurements, we demonstrate that SPR is more suitable for quantitative analysis of DNA binding amount, which is essential for interfacial DNA probe density control and for the analysis of its effect on hybridization efficiency and kinetics. Although the QCM is not quantitative to the same extent as SPR (QCM measures the total mass of the bound DNA molecules together with the associated water), the dissipation factor of the QCM provides a qualitative measure of the viscoelastic properties of DNA films and the conformation of the bound DNA molecules. The complexity in mass measurement does not impair QCM's potential for a kinetic evaluation of the hybridization processes. For quantification of target DNA, the biotin-DNA modified SPR and QCM sensors are exposed to target DNA with increasing concentration. The plots of SPR/QCM signals versus target DNA concentration show that water entrapment between DNA strands make the QCM sensitivity for the hybridization assay well comparable with that of the SPR, although the intrinsic mass sensitivity of the 5 MHz QCM is approximately 20 times lower.     

Impact of spacers on the hybridization efficiency of mixed self-assembled DNA/alkanethiol films

The immobilization of DNA strands is an essential step in the development of any DNA biosensor. Self-assembled mixed DNA/alkanethiol films are often used for coupling DNA probes covalently to the sensor surface. Although this strategy is well accepted, the effect of introducing a spacer molecule to increase the distance between the specific DNA sequence and the surface has rarely been assessed. The major goal of this work was to evaluate a number of such spacers and to assess their impact on for example the sensitivity and the reproducibility. Besides the commonly used mercaptohexyl (C(6)) spacer, a longer mercapto-undecyl (C(11)) spacer was selected. The combination of both spacers with tri(ethylene)glycol (TEG) and hexa(ethylene)glycol (HEG) was studied as well. The effect of the different spacers on the immobilization degree as well as on the consecutive hybridization was studied using surface plasmon resonance (SPR). When using the longer C(11) spacer the mixed DNA/alkanethiol films were found to be more densely packed. Further hybridization studies have indicated that C(11) modified probes improve the sensitivity, the corresponding detection limit as well as the reproducibility. In addition two different immobilization pathways, i.e. flow vs. diffusion controlled, were compared with respect to the hybridization efficiency. These data suggest that a flow-assisted approach is beneficial for DNA immobilization and hybridization events. In conclusion, this work demonstrates the considerable impact of spacers on the biosensor performance but also shows the importance of a flow-assisted immobilization approach.     

Up-converting phosphor reporters for nucleic acid microarrays

An important application of robotically spotted DNA microarrays is the monitoring of RNA expression levels. A clear limitation of this technology is the relatively large amount of RNA that is required per hybridization as a result of low hybridization efficiency and limiting detection sensitivity provided by conventional fluorescent reporters. We have used a recently introduced luminescent reporter technology, called UPT (up-converting phosphor technology). Down-converting phosphors have been applied before to detect nucleic acids on filters using time-resolved fluorometry. The unique feature of the phosphor particles (size 0.4 microm) used here is that they emit visible light when illuminated with infrared (IR) light (980 nm) as a result of a phenomenon called up-conversion. Because neither support material of microarrays nor biomolecules possess up-conversion properties, an enhanced image contrast is expected when these nonfading phosphor particles are applied to detect nucleic acid hybrids on microarrays. Comparison of the UPT reporter to cyanin 5 (Cy5) in a low-complexity model system showed a two order of maginitude linear relationship between phosphor luminescence and target concentration and resulted in an excellent correlation between the two reporter systems for variable target concentrations (R2 = 0.95). However, UPT proved to be superior in sensitivity, even though a wide-field microscope equipped with a xenon lamp was used. This higher sensitivity was demonstrated by complementary DNA (cDNA) microarray hybridizations using cDNAs for housekeeping genes as probes and complex cDNA as target. These results suggest that a UPT reporter technology in combination with a dedicated IR laser array-scanner holds significant promise for various microarray applications.     

Fluorescence in Situ Hybridization (FISH): DNA Probe Production and Hybridization Criteria

We describe methods for the production of fluorescence in situ hybridization (FISH) probes and the utilization of these probes for the detection of complementary DNA sequences with accuracy and sensitivity for application in both basic research and clinical diagnosis. Due to the frequent use of FISH in many laboratories, it is important to apply the most convenient and reproducible approach. This review describes some of the most recent techniques, and includes versatile, effective and simple methods of probe production and fluorescence in situ hybridization. We also describe methods for the production of region-specific and chromosome-specific DNA probes and hybridization techniques for the visualization of these probes.