scholarly journals Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes

1988 ◽  
Vol 251 (3) ◽  
pp. 935-938 ◽  
Author(s):  
A H al-Hakim ◽  
R Hull
Keyword(s):  
Nucleic Acid ◽  
Blot Hybridization ◽  
Nucleic Acid Hybridization ◽  
Cross Linking ◽  
Dot Blot ◽  
Hybridization Probes ◽  
Target Dna ◽  
Amino Derivatives ◽  
Biotin Molecule ◽  
Long Chain

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.

scholarly journals Penggunaan Pelacak DNA untuk Deteksi Papaya ringspot virus dengan Metode Hibridisasi Asam Nukleat

2018 ◽  
Vol 14 (3) ◽  
pp. 89
Author(s):  
Irsan Nuhantoro ◽  
Sri Hendrastuti Hidayat ◽  
Kikin Hamzah Mutaqin
Keyword(s):  
Nucleic Acid ◽  
Detection Method ◽  
Blot Hybridization ◽  
High Specificity ◽  
Dna Probe ◽  
Ringspot Virus ◽  
Nucleic Acid Hybridization ◽  
Disease Spread ◽  
Papaya Ringspot Virus ◽  
Dot Blot

Use of DNA Probe for Detection of Papaya ringspot virus Using Nucleic Acid Hybridization MethodPapaya ringspot caused by Papaya ringspot virus (PRSV) is one of the most destructive diseases of papaya. The disease had not been found in Indonesia, until disease outbreak in Nangroe Aceh Darussalam was reported in 2012. Since then, the disease spread rapidly in most papaya growing areas in Sumatera, Java and Bali. Papaya ringspot virus (PRSV) is generally detected using serological or polymerase chain reaction methods. Improvement in detection method is necessary to facilitate a more reliable tool for controlling the spread of PRSV. The aim of the research was to construct DNA probe for development of detection method based on nucleic acid hybridization. Molecular characterization based on HCPro gene sequence indicated high homology (97.88 to 99.05%) among PRSV isolates from Boyolali (Central Java), Medan (North Sumatera), Sleman (Yogyakarta) and Tabanan (Bali). Two DNA clones of HCPro gene were selected for probe construction and the probes were then labeled using PCR DIG-dioxigenin. Optimization of nucleic acid dot blot hybridization method to achieve strongest positive reaction was developed, i.e. using stringency washes at 1×SSC, 0.1% SDS, incubation at 60 oC for 15’. The DNA probe for PRSV has a high specificity and sensitifity; it could detect PRSV at the lowest concentration of nucleic acid (0.062 µg µL-1).

scholarly journals Evidence for Association of a Viroid with Tapping Panel Dryness Syndrome of Rubber (Hevea brasiliensis)

Plant Disease ◽  
2000 ◽  
Vol 84 (10) ◽  
pp. 1155-1155 ◽  
Author(s):  
P. Ramachandran ◽  
S. Mathur ◽  
L. Francis ◽  
A. Varma ◽  
J. Mathew ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Blot Hybridization ◽  
Potato Spindle Tuber Viroid ◽  
Low Molecular Weight ◽  
Total Nucleic Acid ◽  
Dot Blot ◽  
Dot Blot Hybridization ◽  
Low Salt ◽  
Tapping Panel Dryness

Tapping panel dryness (TPD) is one of the most destructive maladies affecting rubber plantations and is becoming a matter of serious concern. Reduced latex yield leading to total drying of the tapping panel is the obvious symptom. The cause of TPD syndrome is unknown but has been mostly attributed to abiotic causes. In India, the high yielding commercial clone RRII 105 is affected by TPD, leading to enormous losses. We have observed that TPD-affected trees show symptoms of bark scaling, cracking, drying, necrotic streaking, and browning of internal bark leading to the decay of internal tissues. Often prominent abnormal bulges on the lower part of tree trunks occur where the first panel begins to dry. Investigations on TPD-affected rubber samples did not reveal the association of fungus, bacterium, virus, or a protozoan. Total nucleic acid extracts purified from leaf and bark tissues of affected samples and analyzed by polyacrylamide gel electrophoresis under denaturing conditions of low salt and high temperature showed the presence of nucleic acids similar in electrophoretic mobility to low molecular weight (LMW) RNA, of ~359 nucleotides such as potato spindle tuber viroid (PSTVd). The LMW nucleic acid detected from TPD-affected samples was found to be RNA based on its sensitivity to RNase and insensitivity to DNase, phenol, and heat treatments. The LMW RNA was purified and cloned in a pUC 19-derived vector by using primers specific to PSTVd (1). The cloned DNA, when random labeled and used as probe reacted specifically to nucleic acid extracts from TPD-affected rubber trees but not from healthy tissue in dot-blot hybridization assays. Based on the above findings, a viroid etiology for TPD syndrome is proposed. Reference: (1) R. A. Owens, A. T. Candresse, and T. O. Diener. Virology 175:238, 1990.

Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

1990 ◽  
Vol 36 (9) ◽  
pp. 664-669 ◽  
Author(s):  
David R. Preston ◽  
G. Rasul Chaudhry ◽  
Samuel R. Farrah
Keyword(s):  
Tissue Culture ◽  
Nucleic Acid ◽  
Environmental Samples ◽  
Nucleic Acid Hybridization ◽  
Dot Blot ◽  
Polio Virus ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Hybridization Procedure ◽  
Great Specificity

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage [Formula: see text]. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples. Key words: nucleic acid hybridization, polio virus, water, dot blot.

scholarly journals Expanding detection windows for discriminating single nucleotide variants using rationally designed DNA equalizer probes

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Guan A. Wang ◽  
Xiaoyu Xie ◽  
Hayam Mansour ◽  
Fangfang Chen ◽  
Gabriela Matamoros ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rural Areas ◽  
Simultaneous Detection ◽  
Dna Nanotechnology ◽  
Quantitative Relationship ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Hybridization ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Hybridization Probes

Abstract Combining experimental and simulation strategies to facilitate the design and operation of nucleic acid hybridization probes are highly important to both fundamental DNA nanotechnology and diverse biological/biomedical applications. Herein, we introduce a DNA equalizer gate (DEG) approach, a class of simulation-guided nucleic acid hybridization probes that drastically expand detection windows for discriminating single nucleotide variants in double-stranded DNA (dsDNA) via the user-definable transformation of the quantitative relationship between the detection signal and target concentrations. A thermodynamic-driven theoretical model was also developed, which quantitatively simulates and predicts the performance of DEG. The effectiveness of DEG for expanding detection windows and improving sequence selectivity was demonstrated both in silico and experimentally. As DEG acts directly on dsDNA, it is readily adaptable to nucleic acid amplification techniques, such as polymerase chain reaction (PCR). The practical usefulness of DEG was demonstrated through the simultaneous detection of infections and the screening of drug-resistance in clinical parasitic worm samples collected from rural areas of Honduras.

Time-resolved fluorescence detection of enzyme-amplified lanthanide luminescence for nucleic acid hybridization assays

1991 ◽  
Vol 37 (9) ◽  
pp. 1506-1512 ◽  
Author(s):  
E F Templeton ◽  
H E Wong ◽  
R A Evangelista ◽  
T Granger ◽  
A Pollak
Keyword(s):  
Alkaline Phosphatase ◽  
Nucleic Acid ◽  
Fluorescence Detection ◽  
Dna Hybridization ◽  
Nucleic Acid Hybridization ◽  
Time Resolved Fluorescence ◽  
Dot Blot ◽  
Time Resolved ◽  
Lanthanide Luminescence ◽  
Hybridization Assays

Abstract A new nonisotopic detection method based on time-resolved fluorescence for nucleic acid hybridization assays with alkaline phosphatase labels has been developed: enzyme-amplified lanthanide luminescence (EALL). EALL combines the amplification of an enzyme label with the sensitivity and background elimination of time-resolved fluorescence detection of lanthanide ion luminescence. The detection system for alkaline phosphatase makes use of a phosphorylated salicylic acid derivative that, upon dephosphorylation, gives a product capable of forming a luminescent terbium chelate. We demonstrate DNA hybridization assays by using two substrates, one for membrane and one for solution-based formats. Using the substrate that produces a more adhesive product allows performance of dot-blot and Southern blot assays on nylon membranes; results can be recorded with a time-resolved photographic camera system, or with an ultraviolet transilluminator-based system. Less than 4 pg of target sequence can be detected in a dot-blot assay after incubation with substrate for 2-4 h. DNA microwell-plate hybridization assays with the more soluble substrate/product pair can be quantified with time-resolved fluorescence plate readers, giving a similar detection sensitivity. EALL is thus a practical time-resolved fluorescence-based alternative to other detection systems for DNA hybridization assays.

Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes

2006 ◽  
Vol 363 (1-2) ◽  
pp. 48-60 ◽  
Author(s):  
Salvatore A.E. Marras ◽  
Sanjay Tyagi ◽  
Fred Russell Kramer
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Molecular Beacons ◽  
Nucleic Acid Hybridization ◽  
Hybridization Probes
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