scholarly journals Cellular microRNA detection with miRacles: microRNA- activated conditional looping of engineered switches

2019 ◽  
Vol 5 (3) ◽  
pp. eaau9443 ◽  
Author(s):  
Arun Richard Chandrasekaran ◽  
Molly MacIsaac ◽  
Paromita Dey ◽  
Oksana Levchenko ◽  
Lifeng Zhou ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Northern Blotting ◽  
Single Step ◽  
Single Nucleotide ◽  
Disease Biomarkers ◽  
Microrna Detection ◽  
Detection Assay ◽  
Polymerase Chain ◽  
Regulatory Rnas ◽  
Nucleotide Specificity

MicroRNAs are short noncoding regulatory RNAs that are increasingly used as disease biomarkers. Detection of microRNAs can be arduous and expensive and often requires amplification, labeling, or radioactive probes. Here, we report a single-step, nonenzymatic microRNA detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNA-activated conditional looping of engineered switches), our assay has subattomole sensitivity and single-nucleotide specificity using an agarose gel electrophoresis readout. We detect cellular microRNAs from nanogram-scale RNA extracts of differentiating muscle cells and multiplex our detection for several microRNAs from one biological sample. We demonstrate 1-hour detection without expensive equipment or reagents, making this assay a compelling alternative to quantitative polymerase chain reaction and Northern blotting.

scholarly journals The expression and mutation of <i>BMPR1B</i> and its association with litter size in small-tail Han sheep (<i>Ovis aries</i>)

2021 ◽  
Vol 64 (1) ◽  
pp. 211-221
Author(s):  
Yu-Liang Wen ◽  
Xiao-Fei Guo ◽  
Lin Ma ◽  
Xiao-Sheng Zhang ◽  
Jin-Long Zhang ◽  
...  
Keyword(s):  
Litter Size ◽  
Quantitative Polymerase Chain Reaction ◽  
Follicular Development ◽  
Follicular Phase ◽  
Ovis Aries ◽  
Negative Effects ◽  
Single Nucleotide ◽  
Ovarian Granulosa Cell ◽  
Polymerase Chain ◽  
Genotype And Allele Frequencies

Abstract. Previous studies have shown that BMPR1B promotes follicular development and ovarian granulosa cell proliferation, thereby affecting ovulation in mammals. In this study, the expression and polymorphism of the BMPR1B gene associated with litter size in small-tail Han (STH) sheep were determined. The expression of BMPR1B was detected in 14 tissues of STH sheep during the follicular phase as well as in the hypothalamic–pituitary–gonadal (HPG) axis of monotocous and polytocous STH sheep during the follicular and luteal phases using quantitative polymerase chain reaction (qPCR). Sequenom MassARRAY® single nucleotide polymorphism (SNP) technology was also used to detect the polymorphism of SNPs in seven sheep breeds. Here, BMPR1B was highly expressed in hypothalamus, ovary, uterus, and oviduct tissue during the follicular phase, and BMPR1B was expressed significantly more in the hypothalamus of polytocous ewes than in monotocous ewes during both the follicular and luteal phases (P<0.05). For genotyping, we found that genotype and allele frequencies of three loci of the BMPR1B gene were extremely significantly different (P<0.01) between the monotocous and polytocous groups. Association analysis results showed that the g.29380965A>G locus had significant negative effects on the litter size of STH sheep, and the combination of g.29380965A>G and FecB (Fec – fecundity and B – Booroola; A746G) at the BMPR1B gene showed that the litter size of AG–GG, AA–GG, and GG–GG genotypes was significantly higher compared with other genotypes (P<0.05). This is the first study to find a new molecular marker affecting litter size and to systematically analyze the expression of BMPR1B in different fecundity and physiological periods of STH sheep.

Multiplex SNP genotyping in whole blood using an integrated microfluidic lab-on-a-chip

Lab on a Chip ◽  
2016 ◽  
Vol 16 (20) ◽  
pp. 4012-4019 ◽  
Author(s):  
L. Zhang ◽  
Q. Cai ◽  
R. S. Wiederkehr ◽  
M. Fauvart ◽  
P. Fiorini ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Cross Flow ◽  
Lab On A Chip ◽  
Nucleotide Polymorphisms ◽  
Chain Reaction ◽  
Single Nucleotide ◽  
Blood Lysis ◽  
Polymerase Chain ◽  
Silicon Based

We present a silicon-based integrated microsystem combining a blood lysis chamber, a cross-flow filter, a T-junction mixer, and a microreactor for quantitative polymerase chain reaction. The detection of multiple single nucleotide polymorphisms was demonstrated in the system from human blood.

scholarly journals MRPrimerW2: an enhanced tool for rapid design of valid high-quality primers with multiple search modes for qPCR experiments

2019 ◽  
Vol 47 (W1) ◽  
pp. W614-W622 ◽  
Author(s):  
Hajin Jeon ◽  
Jeongmin Bae ◽  
Sang-Hyun Hwang ◽  
Kyu-Young Whang ◽  
Hyun-Seob Lee ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Free Access ◽  
Target Sequence ◽  
Single Pair ◽  
High Quality ◽  
Single Nucleotide ◽  
Complete Set ◽  
Polymerase Chain ◽  
Target Sequences ◽  
Search Modes

Abstract For the best results in quantitative polymerase chain reaction (qPCR) experiments, it is essential to design high-quality primers considering a multitude of constraints and the purpose of experiments. The constraints include many filtering constraints, homology test on a huge number of off-target sequences, the same constraints for batch design of primers, exon spanning, and avoiding single nucleotide polymorphism (SNP) sites. The target sequences are either in database or given as FASTA sequences, and the experiment is for amplifying either each target sequence with each corresponding primer pairs designed under the same constraints or all target sequences with a single pair of primers. Many websites have been proposed, but none of them including our previous MRPrimerW fulfilled all the above features. Here, we describe the MRPrimerW2, the update version of MRPrimerW, which fulfils all the features by maintaining the advantages of MRPrimerW in terms of the kinds and sizes of databases for valid primers and the number of search modes. To achieve it, we exploited GPU computation and a disk-based key-value store using PCIe SSD. The complete set of 3 509 244 680 valid primers of MRPrimerW2 covers 99% of nine important organisms in an exhaustive manner. Free access: http://MRPrimerW2.com

scholarly journals Chromosome 22q11 deletions, velo-cardio-facial syndrome and early-onset psychosis

2003 ◽  
Vol 183 (5) ◽  
pp. 409-413 ◽  
Author(s):  
D. Ivanov ◽  
G. Kirov ◽  
N. Norton ◽  
H. J. Williams ◽  
N. M. Williams ◽  
...  
Keyword(s):  
Early Onset ◽  
Quantitative Polymerase Chain Reaction ◽  
Age At Onset ◽  
Childhood Onset ◽  
Single Nucleotide ◽  
Chromosome 22Q11 ◽  
Childhood Onset Schizophrenia ◽  
Early Onset Psychosis ◽  
Polymerase Chain ◽  
Velo Cardio Facial Syndrome

BackgroundVelo-cardio-facial syndrome (VCFS) is associated with interstitial deletions of chromosome 22q11. About 30% of patients with VCFS have psychosis, and the rate of these deletions in schizophrenia has been reported to be about 1%. Even higher rates of VCFS deletions have been reported for childhood-onset schizophrenia.AimsTo test the hypothesis that there is an increased rate of VCFS among patients with early-onset psychosis (age at onset < 18 years). We screened 192 early-onset patients and 329 patients with adult-onset schizophrenia.MethodWe genotyped the patients and 444 healthy controls for hemizygosity of five microsatellite markers and one single nucleotide polymorphism that map to the 22q11-deleted region.ResultsOne patient had a VCFS deletion, confirmed with semi-quantitative polymerase chain reaction. None of the controls showed a pattern of genotypes consistent with hemizygosity.ConclusionsVCFS may be less frequent among patients with psychosis than previously suggested; this rate is not increased among early-onset patients.

scholarly journals The Rapid Antigen Detection Test for SARS-CoV-2 Underestimates the Identification of COVID-19 Positive Cases and Compromises the Diagnosis of the SARS-CoV-2 (K417N/T, E484K, and N501Y) Variants

2022 ◽  
Vol 9 ◽  
Author(s):  
Carlos Barrera-Avalos ◽  
Roberto Luraschi ◽  
Eva Vallejos-Vidal ◽  
Andrea Mella-Torres ◽  
Felipe Hernández ◽  
...  
Keyword(s):  
Detection Efficiency ◽  
Quantitative Polymerase Chain Reaction ◽  
Detection Sensitivity ◽  
Nucleotide Polymorphisms ◽  
False Negatives ◽  
Single Nucleotide ◽  
Rapid Antigen Detection Test ◽  
Current Scenario ◽  
Variant Detection ◽  
Polymerase Chain

Timely detection of severe acute respiratory syndrome due to coronavirus 2 (SARS-CoV-2) by reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been the gold- strategy for identifying positive cases during the current pandemic. However, faster and less expensive methodologies are also applied for the massive diagnosis of COVID-19. In this way, the rapid antigen test (RAT) is widely used. However, it is necessary to evaluate its detection efficiency considering the current pandemic context with the circulation of new viral variants. In this study, we evaluated the sensitivity and specificity of RAT (SD BIOSENSOR, South Korea), widely used for testing and SARS-CoV-2 diagnosis in Santiago of Chile. The RAT showed a 90% (amplification range of 20 ≤ Cq &lt;25) and 10% (amplification range of 25 ≤ Cq &lt;30) of positive SARS-CoV-2 cases identified previously by RT-qPCR. Importantly, a 0% detection was obtained for samples within a Cq value&gt;30. In SARS-CoV-2 variant detection, RAT had a 42.8% detection sensitivity in samples with RT-qPCR amplification range 20 ≤ Cq &lt;25 containing the single nucleotide polymorphisms (SNP) K417N/T, N501Y and E484K, associated with beta or gamma SARS-CoV-2 variants. This study alerts for the special attention that must be paid for the use of RAT at a massive diagnosis level, especially in the current scenario of appearance of several new SARS-CoV-2 variants which could generate false negatives and the compromise of possible viral outbreaks.

scholarly journals Detection of cellular microRNAs with programmable DNA nanoswitches

2018 ◽  
Author(s):  
Arun Richard Chandrasekaran ◽  
Molly MacIsaac ◽  
Paromita Dey ◽  
Oksana Levchenko ◽  
Lifeng Zhou ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Biological Sample ◽  
Medical Diagnostics ◽  
Northern Blotting ◽  
Single Step ◽  
Detection Methods ◽  
Biological Research ◽  
Clinical Tool ◽  
Single Nucleotide ◽  
Detection Techniques

AbstractMicroRNAs are short non-coding regulatory RNAs that are increasingly used as disease biomarkers. Detection of microRNAs can be arduous and expensive, and often requires amplification, labeling, or radioactive probes. Here we report a single-step, non-enzymatic detection assay using conformationally responsive DNA nanoswitches. Termed miRacles (microRNAactivatedconditionallooping ofengineeredswitches), our assay has sub-attomole sensitivity and single-nucleotide specificity using an agarose gel electrophoresis readout. We detect cellular microRNAs from nanogram-scale RNA extracts of differentiating muscle cells, and demonstrate multiplexed detection of several microRNAs from one biological sample. We demonstrate one-hour detection without expensive equipment or reagents, making this assay a compelling alternative to qPCR and Northern blotting.Significance statementDetection of microRNAs play a key role in biological research and medical diagnostics, and current detection methods are expensive and require sophisticated processes. We presentmicroRNAactivatedconditionallooping ofengineeredswitches (miRacles), a mix-and-read strategy that is based on conformational changes of DNA nanoswitches upon binding a target microRNA. MiRacles has a sensitivity of ∼4 copies/cell and specificity of a single nucleotide, and can be performed in one hour at a fraction of the cost of traditional microRNA detection techniques. Our method can also be multiplexed to detect multiple microRNAs from one biological sample. The minimalistic miRacles assay has immediate application in biomedical research and longer term potential as a clinical tool.

Glyphosate-Resistant Junglerice (Echinochloa colona) from Mississippi and Tennessee: Magnitude and Resistance Mechanisms

Weed Science ◽  
2018 ◽  
Vol 66 (5) ◽  
pp. 603-610 ◽  
Author(s):  
Vijay K. Nandula ◽  
Garret B. Montgomery ◽  
Amaranatha R. Vennapusa ◽  
Mithila Jugulam ◽  
Darci A. Giacomini ◽  
...  
Keyword(s):  
Gene Sequence ◽  
Quantitative Polymerase Chain Reaction ◽  
Point Mutations ◽  
Resistance Index ◽  
Glyphosate Resistance ◽  
Resistance Mechanisms ◽  
Site Mutation ◽  
Single Nucleotide ◽  
Treated Leaf ◽  
Polymerase Chain

AbstractRecently, several incidents of glyphosate failure on junglerice [Echinochloa colona(L.) Link] have been reported in the midsouthern United States, specifically in Mississippi and Tennessee. Research was conducted to measure the magnitude of glyphosate resistance and to determine the mechanism(s) of resistance to glyphosate inE. colonapopulations from Mississippi and Tennessee. ED50(dose required to reduce plant growth by 50%) values for a resistant MSGR4 biotype, a resistant TNGR population, and a known susceptible MSGS population were 0.8, 1.62, and 0.23 kg ae ha−1of glyphosate, respectively. The resistance index calculated from the these ED50values indicated that the MSGR4 biotype and TNGR population were 4- and 7-fold, respectively, resistant to glyphosate relative to the MSGS population. The absorption patterns of [14C]glyphosate in the TNGR and MSGS populations were similar. However, the MSGS population translocated 13% more [14C]glyphosate out of the treated leaf compared with the TNGR population at 48 h after treatment.EPSPSgene sequence analyses of TNGRE. colonaindicated no evidence of any point mutations, but several resistant biotypes, including MSGR4, possessed a single-nucleotide substitution of T for C at codon 106 position, resulting in a proline-to-serine substitution (CCA to TCA). Results from quantitative polymerase chain reaction analyses suggested that there was no amplification of theEPSPSgene in the resistant populations and biotypes. Thus, the mechanism of resistance in the MSGR population (and associated biotypes) is, in part, due to a target-site mutation at the 106 loci of theEPSPSgene, while reduced translocation of glyphosate was found to confer glyphosate resistance in the TNGR population.

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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