Immobilized Oligonucleotide Probes Research Articles

LATERAL FLOW HYBRIDIZATION ASSAY FOR DETERMINATION OF TRANSCRIPTS OF TEM-TYPE BETA-LACTAMASE GENES IN BACTERIA RESISTANT TO ANTIBIOTICS

A technique for lateral ow hybridization analysis of speci c bacterial beta-lactamase mRNAs has been developed, including the preparation of a biotinylated target DNA and its analysis on a test strip with immobilized oligonucleotide probes. When the DNA target and probe structures are complementary, DNA duplexes are formed in the test zone of the strip, which interact with the streptavidin conjugate with gold nanoparticles. The method was used to determine the transcripts of the TEM-type betalactamase genes isolated from the culture of E. coli - producers of the corresponding recombinant betalactamase. It has been shown that singlestranded DNA target of 330 bases was revealed most effectively in lateral ow analysis.

Multi-frequency impedance sensing for detection and sizing of DNA fragments

Electronic biosensors for DNA detection typically utilize immobilized oligonucleotide probes on a signal transducer, which outputs an electronic signal when target molecules bind to probes. However, limitation in probe selectivity and variable levels of non-target material in complex biological samples can lead to nonspecific binding and reduced sensitivity. Here we introduce the integration of 2.8 μm paramagnetic beads with DNA fragments. We apply a custom-made microfluidic chip to detect DNA molecules bound to beads by measuring Impedance Peak Response (IPR) at multiple frequencies. Technical and analytical performance was evaluated using beads containing purified Polymerase Chain Reaction (PCR) products of different lengths (157, 300, 613 bp) with DNA concentration ranging from 0.039 amol to 7.8 fmol. Multi-frequency IPR correlated positively with DNA amounts and was used to calculate a DNA quantification score. The minimum DNA amount of a 300 bp fragment coupled on beads that could be robustly detected was 0.0039 fmol (1.54 fg or 4750 copies/bead). Additionally, our approach allowed distinguishing beads with similar molar concentration DNA fragments of different lengths. Using this impedance sensor, purified PCR products could be analyzed within ten minutes to determine DNA fragment length and quantity based on comparison to a known DNA standard.

Design and Development of Reverse Slot Blot for the Simultaneous Detection of Rare and Regional Specific Mutations in the Beta Globin Gene in Khuzestan Province of Iran.

Beta-thalassemia is the most frequent hemoglobin disorder in Iran resulting from disrupting mutations in the beta globin (HBB) gene that causes decreased or complete absent of beta-globin chains. The screening of beta-thalassemia minor and major individuals and prenatal diagnosis is important for familial planning. Therefore, it is essential, depending on the ethnicity and local frequency of changes, to develop a rapid and accurate method for molecular diagnosis of beta-thalassemia. Here, we developed reverse slot blot (RSB) assay for the simultaneous detection of six common pathogenic changes in the HBB gene (-88, -28, IVSII-745, IVSII-848, Codon 6 [G → A] for HbC, Codon 6 [A → T] for HbS) in the Khuzestan Province of Iran. We designed normal and mutant oligonucleotide probes for each selected mutation and fixed them on positively charged nylon membrane. In the next step, a multiplex-polymerase chain reaction (PCR) performed for the amplification of the entire HBB gene using labelled 5'-biotinylated primers. The PCR products were hybridized to immobilized oligonucleotide probes on the membrane at the appropriate temperature. Finally, we developed the membrane by chemically colorimetric reaction using nitro-blue tetrazolium-5-Bromo-4-chloro-3-indolyl phosphate. For the best probe concentration, we made a serial dilution of probe pairs for each mutation. The optimal probe concentration for each mutation varied from 25 to 50pmol. In the next step, DNA samples from homozygous affecting individuals were subjected for multiple PCR. Hybridization of each PCR products on the nylon membrane with probe pairs revealed specific bands with expected signal intensity without any background. Our designed RSB test is a rapid, sensitive and cost-effective method for screening of regional specific beta-thalassemia mutations in the Khuzestan population of Iran, which might be extended for the detection of any desired pathogenic changes.

Biological microchip for assessing tetracycline-resistance in <i>Neisseria gonorrhoeae</i> clinical isolates in Russian Federation

A total of 399 Neisseria gonorrhoeae clinical isolates collected in different regions of the Russian Federation in 20152017 were analyzed for tetracycline susceptibility and genetic markers of resistance. Drug susceptibility testing was performed by serial dilution method in agar and minimum inhibitory concentration (MIC) was measured according to the Russian “Guidelines for microbial susceptibility testing for antibacterial agents No. 4.2.1890-04”. Tetracycline resistance determinants were studied by using hydrogel microarray with immobilized oligonucleotide probes able to identify a series of chromosomal mutations and detect plasmid tetM gene. Different resistance determinants were found in 193 isolates (48.4%). Mutation in codon 57 in the rpsJ gene (41.2%) was most common that decreases tetracycline affinity to ribosome 30S subunit, mainly due to Val57Met substitution both as a point mutation as well as in combination with others. Mutations in the rpsJ gene were found in strains with the intermediate tetracycline susceptibility. Mutations in the porB gene (lower tetracycline influx) held the se cond place in prevalence pattern (23.1%); the Gly120Lys substitution usually led to emergence of tetracycline resistance either as a point mutation or in combination with other substitutions. Substitutions of Gly120 for other residues (Asp, Asn, and Thr) and Ala121 for Asp, Asn, and Gly had much less effect on resistance level. The –35 delA deletion in the promoter region of mtrR gene (increased expression of MtrC-MtrD-MtrE efflux pump) was observed in 11.3% strains. The tetM gene was found in 27 strains including 17 American and 10 Dutch type tetM determinants. Evolutionary tree was constructed for the tetM genes with the estimation of their homology with similar genes in genera Streptococcus, Enterococcus and Mycoplasma. Mutations in chromosomal genes resulted in increase of tetracycline MIC up to 2–4 mg/L; 4 mg/L MIC was observed in case of simultaneous presence of several mutations. Strains bearing tetM gene-containing plasmid showed extremely high resistance level: MIC ≥ 8 mg/L (64 mg/L for the two samples). Thus, long-lasting withdrawal of tetracycline use for treatment of gonococcal infections in Russia (since 2003) resulted in decreased percentage of resistant strains (including strains with intermediate susceptibility) from 75% down to 45.4%. However, currently tetracycline resistance in Russia remains elevated that is explained by the presence of different resistance determinants in the half of isolates under study.

Voltammetric determination of the Alzheimer's disease-related ApoE 4 gene from unamplified genomic DNA extracts by ferrocene-capped gold nanoparticles.

A sensitive method is described for detection of the apoE 4 gene detection which is important for early diagnosis of Alzheimer's disease. It is based on signal amplification by using ferrocene(Fc) capped gold nanoparticlesmodified withstreptavidin. The immobilized oligonucleotide probe captures complementary apoE 4 gene. This is followed by the specific recognition of theGCGC sequences which are hydrolyzedby the restriction enzyme HhaI. Cleavage only occurs at the complementary apoE 4 duplex, while mismatches prevent enzymatic cleavage. Thus, the apoE 4 sequence can be discriminated against other apoE sequences. Benefitting from amplified signal by Fc-capped nanoparticle/streptavidin and the recognition of HhaI, the detection limit is as low as 0.1 pM of the ApoE 4 gene. Four genomic DNA samples extracted from blood were analyzed for the presence of the apoE 4 gene. The approach presented here will provide viable proof-of-principle for an enzyme-assisted electrochemical assay for the apoE 4 gene in genomic DNAs. Graphical abstract Schematic presentation of amplified voltammetric detection of Alzheimer's Disease-related apoE 4 gene from unamplified genomic DNA extracts via ferrocene capped gold nanoparticle/streptavidin.

A Novel Streptavidin-luciferase Fusion Protein: Preparation, Properties and Application in Hybridization Analysis of DNA.

A streptavidin-luciferase fusion protein comprising the thermostable mutant form of firefly luciferase Luciola mingrelica and minimal core streptavidin was constructed. The streptavidin-luciferase fusion was mainly produced in a tetrameric form with high luciferase and biotin-binding activities. It was shown that fusion has the same Km values for ATP and luciferin and the bioluminescence spectra as initial luciferase. The linear dependence of the bioluminescence signal on the content of the fusion was observed within the range of 10-18 -10-13 mol per well. Successful application of obtained fusion in a biospecific bioluminescence assay based on biotin-streptavidin interactions was demonstrated by the example of a specific DNA hybridization analysis. A DNA hybridization analysis for Escherichia coli cells identification was developed using unique for these cells gadB fragment encoding glutamate decarboxylase. The amplified biotinylated GadB fragments were hybridized with the immobilized oligonucleotide probes; then, the biotin in the DNA duplexes was detected using the streptavidin-luciferase fusion protein. To reach the high sensitivity of the assay, we optimized the conditions of the assay. It was shown that the use of Pluronic for plate modification resulted in a significant reduction in the DNA detection limit which finally was 0.4 ng per well.

Electrochemical detection of 0.6% genetically modified maize MON810 in real flour samples

We demonstrate an approach for the electrochemical detection of genetically modified maize in real maize flour samples by means of square-wave voltammetry (SWV). After labeling the asymmetric PCR-amplified targets with osmium tetroxide bipyridine [OsO4(bipy)], they were hybridized with immobilized oligonucleotide probes on gold electrodes. We could detect the maize genes ivrp and SSIIb in near isogenic maize. The transgene cryIa/b and the MON810 specific fragment were detected in all transgenic maize samples, down to a content of 0.6% of MON810 in mixed samples. While it was possible to detect all sequences in the samples containing 100% near isogenic or respectively transgenic maize after a hybridization time of less than 10min, a hybridization time of 30min was necessary for the detection of the genetic modifications in samples containing only 0.9 to 0.6% of transgenic maize. No significant detection of the transgene cryIa/b or MON810 was possible when only 0.5% of transgenic maize was present in the sample, most likely due to insufficient amplification of the template DNA.

Identification of plant-derived genetically modified organisms in food and feed using a hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in food and feed products. The biochip contained 22 immobilized oligonucleotide probes that were intended for (1) detection of plant DNA, (2) determination of plant species (soybean, maize, potato, and rice), and (3) identification of transgenic elements, including sequences of 35S CaMV, 35S FMV, rice actin gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, and nptII marker genes. The limit of detection was 0.5% for genetically modified (GM) soybean and maize in the analyzed samples. The tests on food and feed products using the developed approach and real-time PCR showed full agreement in determination of transgenic DNA in the samples. The proposed assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of GM component based on the identified transgene.

Microfluidics for the deposition of density gradients of immobilized oligonucleotide probes; developing surfaces that offer spatial control of the stringency of DNA hybridization

A method for the development of continuous density gradients of immobilized oligonucleotide probes (20mer) along the length of microfluidic channels is demonstrated. The development of continuous density gradients was achieved using variable electrokinetic transport of probes in hybrid glass–polydimethylsiloxane microfluidic chips. The probes were terminated with an amine functional group, and were delivered by electrokinetic pumping to the flat glass channel wall after it had been densely coated with covalently immobilized aldehyde groups. This method provided probe immobilization densities ranging from 4.5(±0.8) × 10 13 to 2.5(±0.8) × 10 11 molecules cm −2, with longitudinal dilution and differential mass transport of the injected plug of probes being the primary factors responsible for the gradient of density. The utility of the resulting density gradient of immobilized probes to control the selectivity of hybridization was demonstrated at room temperature by discrimination between a fully complementary oligonucleotide target, and a target strand containing 3 base pair mismatches (3 BPM) based on the spatial pattern of hybridization for sub-picomole quantities of targets. Single nucleotide polymorphism (SNP) discrimination was possible when temperature control was implemented to improve resolution of the mismatch discrimination, allowing SNP discrimination at 35 °C with a contrast ratio of almost 5 to 1.

Analysis of methanotroph community composition using a pmoA-based microbial diagnostic microarray

Microbial diagnostic microarrays (MDMs) are highly parallel hybridization platforms containing multiple sets of immobilized oligonucleotide probes used for parallel detection and identification of many different microorganisms in environmental and clinical samples. Each probe is approximately specific to a given group of organisms. Here we describe the protocol used to develop and validate an MDM method for the semiquantification of a range of functional genes--in this case, particulate methane monooxygenase (pmoA)--and we give an example of its application to the study of the community structure of methanotrophs and functionally related bacteria in the environment. The development and validation of an MDM, following this protocol, takes ∼6 months. The pmoA MDM described in detail comprises 199 probes and addresses ∼50 different species-level clades. An experiment comprising 24 samples can be completed, from DNA extraction to data acquisition, within 3 d (12-13 h bench work).

Single-step covalent immobilization of oligonucleotides onto solid surface

In this communication, we report a simple, single-step method for covalent immobilization of oligonucleotide probes onto an activated polystyrene surface. Polystyrene surface is activated by a photolinker, 1-fluoro-2-nitro-4-azidobenzene (FNAB) in a photochemical reaction by UV light. Amine-modified oligonucleotide probe is covalently immobilized to the activated surface by displacing the labile fluoro group of the activated polystyrene surface by the primary amino group of the aminated oligonucleotide probe. Biotinylated complementary oligonucleotide target is hybridized with the immobilized probe. The hybridized target is detected by binding streptavidin-peroxidase followed by assaying the enzyme for colorimetric signal measurement. The covalently immobilized oligonucleotide probe shows high selectivity in subsequent hybridization processes with the complementary target and clearly discriminates with single-base mismatched oligonucleotide targets. The method can immobilize amine- or thiol-modified oligonucleotide onto FNAB–activated polymer surface. The method could be potentially useful for designing DNA based biosensors or biochips and also in microtiter plate based assay systems in a cost effective manner.

A mixed film composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) brushes to enhance selectivity for detection of single nucleotide polymorphisms

Preliminary studies of mixed films composed of oligonucleotides and poly(2-hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for detection of 3 base-pair mismatched (3 bpm) oligonucleotide targets. Evaluation of selectivity for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been completed. The selectivity was quantitatively determined by considering the sharpness of melt curves and melting temperature differences (Δ T m) for fully complementary targets and SNPs. Stringency conditions were investigated, and it was determined that the selectivity was maximized when a moderate ionic strength was used (0.1–0.6 M). Increases of Δ T m when using mixed films were up to 3-fold larger compared to surfaces containing only immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1 bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces. Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes labeled with Cy3 dye indicated the difference in microenvironment of immobilized oligonucleotides in the presence of PHEMA.

HLA type and risk of alloimmunization in sickle cell disease

Red blood cell (RBC) transfusions are frequently required to treat patients with sickle cell disease (SCD) [1]. One of the most serious complications of repeat transfusion is alloimmunization to RBC antigens [2]. Because human leukocyte antigen (HLA) genes mediate the response to foreign antigens, particular HLA alleles may predispose to the development of alloimmunization in patients with SCD who receive multiple transfusions. We conducted a case-control study to determine if particular HLA alleles are associated with alloimmunization and whether HLA homozygosity influences the risk of developing RBC alloantibodies. High-resolution HLA genotyping was performed on DNA samples from 159 multiply transfused patients with SCD. HLA allele frequencies were compared between alloantibody-positive and alloantibody-negative groups. The HLA-DRB1 * 1503 allele was associated with an increased risk (P = 0.039), while HLA-DRB1 * 0901 conferred protection from alloimmunization (P = 0.008). HLA Class II locus homozygosity was more frequently observed in the alloantibody-negative group (P = 0.01). These preliminary findings suggest that particular HLA-DR81 alleles and overall homozygosity at HLA class II loci are associated with alloimmunization risk in SCD. If confirmed, HLA type may serve as a useful genetic predictor of alloimmunization risk, and permit a targeted approach to the use of phenotypically matched blood.

Optical Imaging of Surface-Immobilized Oligonucleotide Probes on DNA Microarrays Using Liquid Crystals

In this paper, we report a new label-free method for the imaging of immobilized oligonucleotide probes on DNA microarrays. The imaging principle is based on the disruption of orientations of nematic liquid crystals (LCs), 4-cyano-4'-pentylbiphenyl (5CB), by the immobilized oligonucleotides on a surface. Because LCs are birefringent materials, disruption of their orientations by the immobilized oligonucleotides can manifest as optical signals visible to the naked eye. LC cells with two homeotropic boundary conditions, which align 5CB perpendicularly to both surfaces, were developed to deliver a distinct contrast between a dark background and a bright image caused by the immobilized oligonucleotides. This design also allows the quantification of immobilized oligonucleotide concentrations through the interference colors of LCs. The LC-based imaging method has a good signal-to-noise ratio and a clear distinction between positive and negative results and is nondestructive to the immobilized oligonucleotides. These advantages make it a promising means of assessing the quality of DNA microarrays.

Real-time monitoring ofcarbonariusDNA structured biochip by surface plasmon resonance imaging

Surface plasmon resonance imaging (SPRI) studies, performed on a specially designed system exploiting the Kretschmann configuration, have been carried out to develop a DNA sensor for the detection of gene mutations accounting for the analysis of a fungin species which can proliferate especially in cereals, producing toxic compounds such as mycotoxins. The SPRI system has been used in order to study the hybridization process of ssDNA carbonarius probes immobilized onto a bio-functionalized Au surface in order to detect in real time the mutations in a DNA fragment. The SPRI system is a good choice for real-time monitoring of hybridization dynamics on an array of immobilized oligonucleotide probes because of the high sensitivity in characterization of ultra-thin films adsorbed onto gold or other noble metal surfaces. Using this technique, local changes in the reflectivity of a thin metal film describe the hybridization process between the molecules tethered to the surface and those sent in solution in the test chamber. The increase in the greyscale levels of the images (representing the functionalized gold traps) during the hybridization process demonstrated the occurrence of the binding event. The process has been proven to be reversible and specific for the investigated probes, since no signal has been detected in the presence of a negative control which is a non-complementary target.

Light Induced Surface Corrosion of Gallium Arsenide for Immobilization of Oligonucleotide Probes

The potential to modify gallium arsenide (GaAs) surfaces with organic molecules provides possibilities to develop biosensors. Thiol‐terminated oligonucleotides have been self assembled on GaAs substrates. The homogeneity and surface morphology of oligonucleotide films were investigated using atomic force microscopy (AFM) and fluorescence microscopy. X‐ray photoelectron spectroscopy (XPS), and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) were used to determine the surface elemental composition. When in contact with water, the surface of GaAs tended to reorganize. Elemental mapping indicated that the surface was passivated by thiol linked DNA probes. Extended contact with water increased the extent of oxidation and the quantity of immobilized oligonucleotide probes.

Estrogen receptor alpha genotype is associated with a reduced prevalence of radiographic hip osteoarthritis in elderly Caucasian women

This study evaluated the association between polymorphisms in the estrogen receptor (ER) alpha gene (ESR1) and prevalent and incident radiographic hip osteoarthritis (RHOA) in a large, well-defined prospective cohort of elderly Caucasian women. Prevalent and incident RHOA was evaluated from all available pelvis X-rays obtained from the Study of Osteoporotic Fractures at baseline and after a mean of 8.3 years. Evaluable DNA samples were available from 4746 of these subjects. RHOA cases were defined by published methods. The ESR1 polymorphisms at intron I (Pvu II for a T/C substitution and Xba I for an A/G substitution) were genotyped in the context of a multiplex polymerase chain reaction (PCR) amplification followed by allele-specific single nucleotide polymorphism (SNP) detection with immobilized oligonucleotide probes in linear arrays. Multiple logistic regression was performed to estimate odds ratios (ORs) and 95% confidence intervals (95% CI) associated with the T/C and A/G polymorphisms. RHOA was present in 12.1% of subjects, of whom 325 had joint space narrowing (JSN) score > or =3 and 130 had an osteophyte score > or =2 and JSN score > or =2. There was a significant reduction in the odds of prevalent RHOA for individuals with the C/C compared to T/T genotype at the Pvu II site with an OR of 0.71 (95% CI: 0.55-0.92) (P=0.01). Adjustments for age, weight, height, hip Bone mineral density (BMD) and estrogen use did not alter the relationship between the C/C genotype and reduced risk of RHOA, with an OR of 0.71 (95% CI: 0.54-0.94) (P=0.01). The risk of incident RHOA was reduced for the Pvu II C/C compared to the T/T genotype (P=0.11). Also, the reduced risk of incident RHOA in C/C subjects varied by estrogen use. There was no association between the Xba I G/G or G/A genotypes and RHOA with OR of 0.82 (95% CI: 0.61-1.10) (P=0.19) compared to women with A/A genotype. We conclude that the C/C genotype of the ER alpha Pvu II polymorphism was associated with a modestly reduced risk of prevalent and incident RHOA in elderly Caucasian women. Additional work is required to understand how the intron I ESR1 polymorphism may alter joint degeneration.

Application of probes having 2′-deoxyinosine for typing of single nucleotide polymorphisms (SNPs) using DNA microarray

The principle of hybridizing oligonucleotides is employed with single nucleotide polymorphisms (SNPs) typing, but that typing frequently failed. We investigated the SNPs typing of multi-alleles on a chip. Two bases adjacent to the SNPs on the immobilized oligonucleotide probes were substituted for the 2′-deoxyinosine (dI) residues. The biotinylated probes containing the partial sequence of CYP2C9, CYP2C19, GLC1A and ApoE alleles were applied to the streptavidin-embedded plasma-polymerized (PP) DNA microarrays. For the probes containing dI, up to a 2–5-fold difference in the match to a mismatch oligonucleotide fluorescence intensity was found as the probes without dI were 1.5-fold or less. According to the substitution for dI, the hybridization accuracy was enhanced, and then several single-base mismatches were detected on a chip.

Transcription in response to physical stress—clues to the molecular mechanisms of exercise‐induced asthma

To clarify stress-induced immunological reactions and molecular events during exercise and the potential relevance to exercise-induced bronchoconstriction, transcriptional responses to standardized physical stress were determined. Six healthy, young volunteers underwent an endurance exercise of 90% of their individual anaerobic threshold for 90 min. Time-dependent alterations in the expression pattern of leukocytes from healthy, trained subjects were analyzed by DNA microarrays before and 2 h and 6 h after exercise. Starting out from a large collection of cDNA library clones comprising more than 70,000 human expressed sequence tags, we selected, designed, and immobilized oligonucleotide probes (60-70mers) for transcripts of 5000 stress- and inflammation-relevant genes. Exercise-induced stress provoked changes in the expression of 433 gene activities 2 h and/or 6 h after exercise, which could be grouped into six clusters. The most prominent feature was an enhanced transcription of two genes, coding for 5-lipoxygenase (ALOX5) and ALOX5-activating protein. Moreover, enhanced levels of leukotriene B4 (LTB4) and LTC4 (P<0.05) were detected in plasma after exercise. Our data demonstrate that exercise alters the activities of a distinct number of genes. In particular, they possibly provide novel insights into the molecular mechanisms of exercise-induced bronchoconstriction and suggest that enhanced transcription of ALOX5 and its activating protein together with a present predisposition of the subject critically contribute to exercise-induced asthma.

Alteration of the selectivity of hybridization of immobilized oligonucleotide probes by co-immobilization with charged oligomers of ethylene glycol

A significant challenge exists in the creation of an environment for immobilized probe oligonucleotides that offer good structural regularity and reproducibility, where nearest neighbour interactions provide for control of selectivity, yet where the degree of hybridization does not alter nearest neighbour interactions. This new work explores whether a “matrix isolation” method will produce the desired environment for the probe molecules. The DNA oligonucleotide probes are polyelectrolytes with charged backbones and significant flexibility. It is possible to isolate the probe molecules by surrounding each, on average, with a sheath of immobilized oligomer that is not based on complementary nucleic acid, yet that is a polyelectrolyte in order to control the surface density and charge within the mixed film. Preliminary work investigates a mixture of dT 20 as the probe oligonucleotide, and a 20-mer oligomer primarily containing ethylene glycol phosphate, as a matrix isolation material in a 1:20 mole ratio, respectively. Melt temperature ( T m) measurements indicate that the thermodynamic stability of the probe molecules can be adjusted using the oligomer matrix to achieve lower T m values by up to 5 °C, with full retention of selectivity for discrimination of single base pair mismatches even under conditions where the probes at a surface are saturated with complementary target.