ABSTRACT
Anti-DNA antibodies are known to be classical serological hallmarks of systemic lupus erythematosus (SLE). In addition to high-affinity antibodies, the autoantibody pool also contains natural catalytic anti-DNA antibodies that recognize and hydrolyze DNA. However, the specificity of such antibodies is uncertain. In addition, DNA binding to a surface such as the cell membrane, can also affect its recognition by antibodies. Here, we analyzed the hydrolysis of short oligodeoxyribonucleotides (ODNs) immobilized on the microarray surface and in solution by catalytic anti-DNA antibodies from SLE patients. It has been shown that IgG antibodies from SLE patients hydrolyze ODNs more effectively both in solution and on the surface, compared to IgG from healthy individuals. The data obtained indicate a more efficient hydrolysis of ODNs in solution than immobilized ODNs on the surface. In addition, differences in the specificity of recognition and hydrolysis of certain ODNs by anti-DNA antibodies were revealed, indicating the formation of autoantibodies to specific DNA motifs in SLE. The data obtained expand our understanding of the role of anti-DNA antibodies in SLE. Differences in the recognition and hydrolysis of surface-tethered and dissolved ODNs need to be considered in DNA microarray applications.
ABSTRACT
A variant of the hybridization oligonucleotide microarray, utilizing the principle of many probes-one spot (MPOS-microarrays), is proposed. A case study based on Orthopoxviruses (Variola, Monkeypox, and Ectromelia viruses) demonstrates a considerable increase in the fluorescence signal (up to 100-fold) when several oligonucleotide probes are printed to one spot. Moreover, the specificity of detection also increases (almost 1000-fold), allowing the use of probes that individually lack such high specificity. The optimal probes have a Tm of 32-37 °C and length of 13-15 bases. We suggest that the high specificity and sensitivity of the MPOS-microarray is a result of cooperativity of DNA binding with all probes immobilized in the spot. This variant of DNA detection can be useful for designing biosensors, tools for point-of-care (POC) diagnostics, microbial ecology, analysis of clustered regularly interspaced short palindromic repeats (CRISPR), and others. Graphical abstract á .
Subject(s)
DNA, Viral/analysis , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis/instrumentation , Orthopoxvirus/genetics , Base Sequence , DNA, Viral/genetics , Equipment Design , Immobilized Nucleic Acids/chemistry , Immobilized Nucleic Acids/genetics , Oligonucleotide Probes/chemistry , Oligonucleotide Probes/genetics , Orthopoxvirus/chemistry , Poxviridae Infections/virologyABSTRACT
The fluorescence quenchers BHQ1 and BHQ2 can be modified by trace amounts of ammonium persulfate, used for initiating gel polymerization, in electrophoretic purification of TaqMan probes using a denaturing polyacrylamide gel. The case study of BHQ1 quencher has demonstrated that a Boyland-Sims reaction proceeds in the presence of ammonium persulfate to give the corresponding sulfate. The absorption maximum of the resulting quencher shifts to the short-wavelength region relative to the absorption maximum of the initial BHQ1. The TaqMan probe containing such a quencher is less efficient as compared with the probe carrying an unmodified BHQ1. The presence of fluorescein in TaqMan probe plays decisive role in this transformation: the quencher modification proceeds at a considerably lower rate when the fluorescein is absent or replaced with a rhodamine dye (for example, R6G). It is assumed that the observed reaction can take place in two ways-both in darkness and in the reaction of the quencher in an excited state due to energy transfer from the fluorophore irradiated by light.
Subject(s)
Electrophoresis , Fluorescein/chemistry , Fluorescein/isolation & purification , Fluorescent Dyes/chemistry , Fluorescent Dyes/isolation & purification , Taq Polymerase/chemistry , Taq Polymerase/metabolismABSTRACT
A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1-H13, H15, H16) and neuraminidase (N1-N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus.
Subject(s)
Influenza A virus/genetics , Molecular Typing/methods , Oligonucleotide Array Sequence Analysis/methods , Algorithms , Animals , Base Sequence , DNA, Complementary/metabolism , Genetic Techniques , Genotype , Hemagglutinins/genetics , Humans , Models, Genetic , Molecular Sequence Data , Neuraminidase/genetics , Oligonucleotide Probes , Phylogeny , Polymerase Chain ReactionABSTRACT
A microarray method was developed for simultaneous detection and identification of six species of Orthopoxvirus (OPV) including Variola, Monkeypox, Cowpox, Camelpox, Vaccinia, and Ectromelia viruses. The method allowed us to discriminate OPV species from varicella-zoster virus (VZV), Herpes Simplex 1 virus (HSV-1), and Herpes Simplex 2 virus (HSV-2) that cause infections with clinical manifestations similar to OPV infections. The nucleotide sequences of the C23L/B29R and the B19R genes identified for 86 and 72 different OPV strains, respectively, were used to design species-specific microarray oligonucleotide probes (oligoprobes). The microarray also contained several oligoprobes selected from the ORF31, US4, and US5 genes of VZV, HSV-1, and HSV-2, respectively. The samples (from HSVs or OPVs) of ssDNAs for analyses were prepared by using asymmetric PCR followed by chemical labeling of ssDNA with Cy3 dye. DNA from 52 samples of various OPV species, two isolates of VZV, two of HSV-1, and three of HSV-2 were tested using the developed microarray assay; all tested viruses were accurately identified. To ensure the robustness of the microarray assay, three additional unrelated variola virus strains with unknown sequences of the C23L/B29R and the B19R genes were tested. In each instance the microarray unambiguously identified them as Variola virus species. The results obtained in this study demonstrated that this new microarray method is a valuable tool for the rapid and accurate detection and differentiation of these important viral pathogens.
