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BMC Genomics Dec 2012DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and...
A model of binding on DNA microarrays: understanding the combined effect of probe synthesis failure, cross-hybridization, DNA fragmentation and other experimental details of affymetrix arrays.
BACKGROUND
DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and for gene expression analysis. These arrays provide large amounts of data. This data is analyzed using statistical methods that quite often discard a large portion of the information. Most of the information that is lost comes from probes that systematically fail across chips and from batch effects. The aim of this study was to develop a comprehensive model for hybridization that predicts probe intensities for Affymetrix arrays and that could provide a basis for improved microarray analysis and probe development. The first part of the model calculates probe binding affinities to all the possible targets in the hybridization solution using the Langmuir isotherm. In the second part of the model we integrate details that are specific to each experiment and contribute to the differences between hybridization in solution and on the microarray. These details include fragmentation, wash stringency, temperature, salt concentration, and scanner settings. Furthermore, the model fits probe synthesis efficiency and target concentration parameters directly to the data. All the parameters used in the model have a well-established physical origin.
RESULTS
For the 302 chips that were analyzed the mean correlation between expected and observed probe intensities was 0.701 with a range of 0.88 to 0.55. All available chips were included in the analysis regardless of the data quality. Our results show that batch effects arise from differences in probe synthesis, scanner settings, wash strength, and target fragmentation. We also show that probe synthesis efficiencies for different nucleotides are not uniform.
CONCLUSIONS
To date this is the most complete model for binding on microarrays. This is the first model that includes both probe synthesis efficiency and hybridization kinetics/cross-hybridization. These two factors are sequence dependent and have a large impact on probe intensity. The results presented here provide novel insight into the effect of probe synthesis errors on Affymetrix microarrays; furthermore, the algorithms developed in this work provide useful tools for the analysis of cross-hybridization, probe synthesis efficiency, fragmentation, wash stringency, temperature, and salt concentration on microarray intensities.
Topics: DNA Fragmentation; DNA Probes; Hybridization, Genetic; Models, Genetic; Oligonucleotide Array Sequence Analysis
PubMed: 23270536
DOI: 10.1186/1471-2164-13-737 -
Angewandte Chemie (International Ed. in... Jul 2021The ability of aptamers to recognize a variety of different molecules has fueled their emergence as recognition agents to probe complex media and cells. Many detection...
The ability of aptamers to recognize a variety of different molecules has fueled their emergence as recognition agents to probe complex media and cells. Many detection strategies require aptamer binding to its target to result in a dramatic change in structure, typically from an unfolded to a folded state. Here, we report a strategy based on forced intercalation (FIT) that increases the scope of aptamer recognition by transducing subtle changes in aptamer structures into fluorescent readouts. By screening a library of green-fluorescent FIT-aptamers whose design is guided by computational modeling, we could identify hits that sense steroids like dehydroepiandrosterone sulfate (DHEAS) down to 1.3 μM with no loss in binding affinity compared to the unmodified aptamer. This enabled us to study DHEAS in clinical serum samples with several advantages over gold standard methods, including rapid readout (<30 min), simple instrumentation (plate-reader), and low sample volumes (10 μL).
Topics: DNA Probes; Fluorescent Dyes; Humans; Steroids
PubMed: 33878237
DOI: 10.1002/anie.202103440 -
Molecules (Basel, Switzerland) Jan 2010In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific...
In this paper we describe an isothermal rolling-circle amplification (RCA) protocol to detect gene point mutations on chips. The method is based on an allele-specific oligonucleotide circularization mediated by a special DNA ligase. The probe is circularized when perfect complementary sequences between the probe oligonucleotide and HIV cDNA gene. Mismatches around the ligation site can prevent probe circularization. The circularized probe (C-probe) can be amplified by rolling circle amplification to generate multimeric singlestranded DNA (ssDNA) under isothermal conditions. There are four sequence regions to bind respectively with fluorescent probe, RCA primer, solid probe and HIV cDNA template in the C-probe which we designed. These ssDNA products are hybridized with fluorescent probes and solid probes which are immobilized on a glass slide composing a regular microarray pattern. The fluorescence signals can be monitored by a scanner in the presence of HIV cDNA templates, whereas the probes cannot be circularized and signal of fluorescence cannot be found. The RCA array has capability of high-throughput detection of the point mutation and the single-nucleotide polymorphism (SNP).The development of C-probe-based technologies offers a promising prospect for situ detection, microarray, molecular diagnosis, single nucleotide polymorphism, and whole genome amplification.
Topics: DNA Probes; DNA, Complementary; Electrophoresis, Agar Gel; HIV; Nucleic Acid Amplification Techniques; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Point Mutation
PubMed: 20335932
DOI: 10.3390/molecules15020619 -
Bioconjugate Chemistry Oct 2012Herein, we report the discovery of a novel DNA probe with a stem-chelate-loop structure, wherein the stability of the probe-target duplex can be modulated lower or...
Herein, we report the discovery of a novel DNA probe with a stem-chelate-loop structure, wherein the stability of the probe-target duplex can be modulated lower or higher using a narrow concentration range of dilute transition metal ions (0.1-10 μM). Oligonucleotide probes containing two terpyridine (TPY) ligands separated by 15 bases of single-stranded DNA, with or without a flanking 5 base self-complementary DNA stem, were tested in thermal transition studies with linear target DNA and varying amounts of ZnCl(2). Without the stem, addition of Zn(2+) resulted only in reversible destabilization of the probe-target duplex, consistent with assembly (up to 1 equiv Zn(2+)) and disassembly (excess Zn(2+)) of the intramolecular Zn(2+)-(TPY)(2) chelate. Surprisingly, probes including both the intramolecular chelate and the stem gave a probe-target duplex that was reversibly destabilized and stabilized upon addition of Zn(2+) by ±5-7 °C, a phenomenon consistent with assembly and then disassembly of the entire stem-Zn(2+)-(TPY)(2) motif, including the DNA stem. Stem-chelate-loop probes containing dipicolylamine (DPA) ligands exhibited no metal-dependent stabilization or destabilization. The stem-Zn(2+)-(TPY)(2) motif is readily introduced with automated synthesis, and may have broad utility in applications where it is desirable to have both upward and downward, reversible metal-dependent control over probe-target stability involving an unmodified DNA target.
Topics: Base Sequence; Chelating Agents; DNA; DNA Probes; Inverted Repeat Sequences; Models, Molecular; Nucleic Acid Conformation; Nucleic Acid Hybridization; Zinc
PubMed: 22989029
DOI: 10.1021/bc3003293 -
Scientific Reports Mar 2014This work proposes the concept of ratiometric electrochemical proximity assay (REPA), which can be used for one-step, highly sensitive and selective detection of...
This work proposes the concept of ratiometric electrochemical proximity assay (REPA), which can be used for one-step, highly sensitive and selective detection of protein. The assay strategy was achieved on a sensing interface that was formed by hybridization of methylene blue (MB)-labeled antibody-DNA probe (MB-DNA1-Ab1) with ferrocene (Fc)-labeled DNA capture probe (Fc-P) modified gold electrode. On the interface the target protein could trigger the formation of immunocomplex between MB-DNA1-Ab1 and detection antibody-DNA probe (Ab2-DNA2) and subsequently the proximity hybridization of DNA1-DNA2, which led to the departure of MB-DNA1-Ab1 from the interface. The remained Fc-P could form a hairpin structure to take Fc group to electrode surface. Therefore, the recognition of target protein to Ab1 and Ab2 resulted in both the "signal-off" of MB and the "signal-on" of Fc for dual-signal electrochemical ratiometric readout. The proposed REPA could be carried out in one-step with 40-min duration and showed a wide detection range from 0.05 to 100 ng/mL with pg/mL limit of detection, displaying great potential for convenient point-of-care testing and commercial application.
Topics: Antibodies; Antigen-Antibody Complex; Biosensing Techniques; Calibration; DNA Probes; Electrochemical Techniques; Electrodes; Ferrous Compounds; Gold; Humans; Immunoglobulin Fc Fragments; Limit of Detection; Male; Metallocenes; Methylene Blue; Prostate-Specific Antigen
PubMed: 24618513
DOI: 10.1038/srep04360 -
BioTechniques Jan 2004Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation... (Comparative Study)
Comparative Study
Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized this latter two-step approach for maximal hybridization signal brightness using DNA probes labeled to varying degrees with different fluorescent dyes. Reverse transcriptase and DNA polymerase 1 efficiently incorporated aa-dUTP into DNA, and adjusting the aa-dUTP:dTTP ratio controlled the degree of substitution. With cDNA probes hybridized to dot blots, probes having approximately eight dyes per 100 bases gave the best sensitivity, irrespective of the dye label. alpha-Satellite probes generated by nick translation and hybridized to human chromosome spreads also showed that probes having approximately eight dyes per 100 bases provided the brightest overall signals. These data demonstrate that this labeling method generates highly sensitive DNA probes that are difficult to obtain by conventional direct incorporation approaches. The technique is inherently consistent and versatile by virtue of the efficient incorporation of primary amines and the reliable chemical labeling reaction.
Topics: Amines; DNA Probes; Fluorescent Dyes; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence; Staining and Labeling
PubMed: 14740493
DOI: 10.2144/04361RR02 -
Journal of Clinical Microbiology May 1995Cysticercosis results from ingestion of the eggs of the tapeworm Taenia solium. Reduction of the incidence of human and swine cysticercosis requires identification and...
Cysticercosis results from ingestion of the eggs of the tapeworm Taenia solium. Reduction of the incidence of human and swine cysticercosis requires identification and treatment of individuals who carry the adult tapeworm. T. solium and Taenia saginata eggs cannot be differentiated on the basis of morphology; thus, in order to improve existing methods for the diagnosis of taeniasis, we have developed highly sensitive, species-specific DNA probes which differentiate T. solium and T. saginata. Recombinant clones containing repetitive DNA sequences which hybridize specifically with genomic DNAs from either species were isolated and characterized. T. solium-specific DNA sequences contained complete and truncated forms of a tandemly repeated 158-bp DNA sequence. An unrelated T. saginata DNA sequence was also characterized and shown to encode a portion of the mitochondrial cytochrome c oxidase I gene. T. solium- and T. saginata-specific DNA probes did not hybridize in dot blot assays either with genomic DNA from the platyhelminths Taenia hydatigena, Taenia pisiformis, Taenia taeniaeformis, Echinococcus granulosus, and Schistosoma mansoni or with genomic DNA from other eukaryotes, including Saccharomyces cerevisiae, Candida albicans, Cryptosporidium parvum, Entamoeba histolytica, Trypanosoma gambiense, Trypanosoma brucei, and Giardia lamblia, Caenorhabditis elegans, and human DNA. By using these T. solium and T. saginata DNA probes, a rapid, highly sensitive and specific dot blot assay for the detection of T. solium eggs was developed.
Topics: Animals; Base Sequence; Cloning, Molecular; Cysticercosis; DNA Probes; DNA, Helminth; DNA, Recombinant; Female; Humans; Molecular Probe Techniques; Molecular Sequence Data; Ovum; Parasitology; Repetitive Sequences, Nucleic Acid; Restriction Mapping; Sensitivity and Specificity; Species Specificity; Taenia; Taeniasis
PubMed: 7615742
DOI: 10.1128/jcm.33.5.1283-1288.1995 -
Canadian Journal of Veterinary Research... Apr 1992Radiometric (RCM) and conventional fecal culture (HEY) and a commercial polymerase chain reaction/DNA probe were evaluated as diagnostic tests for subclinical...
Radiometric (RCM) and conventional fecal culture (HEY) and a commercial polymerase chain reaction/DNA probe were evaluated as diagnostic tests for subclinical paratuberculosis in dairy cattle using fecal specimens from a repository of paratuberculosis specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis, by conventional or radiometric culture, from fecal samples or internal organs of dairy cattle without diarrhea or chronic weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. Among 182 infected cattle, RCM and HEY fecal culture and the DNA probe had test sensitivities of 54.4%, 45.1% and 33.5%, respectively. Fecal samples from only 111 of the M. paratuberculosis-infected cows tested positive by at least one of the three tests and these cows were designated as fecal shedders; the remaining 71 were considered to have prepatent infections. Among the 111 M. paratuberculosis fecal shedders, RCM, HEY and the probe detected the organism in 89.2%, 73.8% and 55.0% of the fecal specimens, respectively. Herd prevalence significantly affected the sensitivity of all three diagnostic tests (p less than 0.05) but only affected the fecal shedder detection efficiency of the DNA probe (p less than 0.01). No positive DNA probe results were found on 100 randomly selected fecal samples from cows in four certified paratuberculosis-free herds, thus the DNA probe was 100% specific. Probe analyses could be performed in 24 h or less. Time to complete the culture-based tests was 12 wk for HEY and 7 wk for RCM.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Cattle; Cattle Diseases; DNA Probes; Evaluation Studies as Topic; Feces; Female; Mycobacterium tuberculosis; Paratuberculosis; Prevalence; Radiometry; Sensitivity and Specificity
PubMed: 1591658
DOI: No ID Found -
Nucleic Acids Research Jul 1989In order to develop non-radioactive oligonucleotide derivatives and to examine their utility as a diagnostic tool, namely as DNA-probe, an enzyme-linked oligonucleotide...
In order to develop non-radioactive oligonucleotide derivatives and to examine their utility as a diagnostic tool, namely as DNA-probe, an enzyme-linked oligonucleotide was synthesized. Oligonucleotide complementary to M13mp8 phage DNA was linked to alkaline phosphatase via a crosslinker and a spacer. M13mp8 phage DNA (single strand) immobilized on the nitrocellulose membrane was hybridized with the enzyme-linked oligonucleotide. The hybrid was detected with three detection methods; (1)colorimetric detection in solution, (2)colorimetric one on membranes, and (3)fluorometric one in solution. Methods(2) and (3) gave high sensitivities to detect as low as several to several tens attomoles of DNA and it was found that those methods with enzyme-linked oligonucleotides are potent for DNA-probe methodology from the viewpoint of automation.
Topics: Chromatography, Gel; Chromatography, High Pressure Liquid; Colorimetry; Cytosine; DNA; DNA Probes; DNA, Viral; Indicators and Reagents; Oligodeoxyribonucleotides; Spectrometry, Fluorescence
PubMed: 2762149
DOI: 10.1093/nar/17.14.5587 -
Journal of Clinical Microbiology May 1989A total of 201 endocervical specimens were obtained from patients with a clinical or epidemiological history suggestive of chlamydial infection. These specimens were... (Comparative Study)
Comparative Study
A total of 201 endocervical specimens were obtained from patients with a clinical or epidemiological history suggestive of chlamydial infection. These specimens were tested by DNA probe (Gen-Probe, San Diego, Calif.) and the IDEIA III (Boots-Celltech, Berkshire, United Kingdom) monoclonal antibody enzyme immunoassay and compared with cell culture for detection of Chlamydia trachomatis. Discrepancies between cell culture and antigen detection methods were resolved by direct fluorescent-antibody testing. In a population with a 17.4% prevalence, the sensitivities and specificities of these assays were 82.8 and 99.4%, respectively, for the DNA probe assay and 97.1 and 98.1%, respectively, for the IDEIA III.
Topics: Cervix Uteri; Chlamydia Infections; Chlamydia trachomatis; DNA Probes; DNA, Bacterial; Female; Genital Diseases, Female; Humans; Immunoenzyme Techniques; Predictive Value of Tests
PubMed: 2663916
DOI: 10.1128/jcm.27.5.826-828.1989