scholarly journals First Characterization of a Biphasic, Switch-like DNA Amplification

2017 ◽  
Author(s):  
Burcu Özay ◽  
Cara M Robertus ◽  
Jackson L Negri ◽  
Stephanie E McCalla
Keyword(s):  
Dna Amplification ◽  
High Gain ◽  
Clinical Diagnostics ◽  
Second Phase ◽  
Binding Domains ◽  
Trigger Loop ◽  
Reaction Acceleration ◽  
Biphasic Reaction ◽  
Dna Template ◽  
Amplification Reaction

ABSTRACTWe report the first DNA amplification chemistry with switch-like characteristics: the chemistry is biphasic, with an expected initial phase followed by an unprecedented high gain burst of product oligonucleotide in a second phase. The first and second phases are separated by a temporary plateau, with the second phase producing 10 to 100 times more product than the first. The reaction is initiated when an oligonucleotide binds and opens a palindromic looped DNA template with two binding domains. Upon loop opening, the oligonucleotide trigger is rapidly amplified through cyclic extension and nicking of the bound trigger. Loop opening and DNA association drive the amplification reaction, such that reaction acceleration in the second phase is correlated with DNA association thermodynamics. Without a palindromic sequence, the chemistry resembles the exponential amplification reaction (EXPAR). EXPAR terminates at the initial plateau, revealing a previously unknown phenomenon that causes early reaction cessation in this popular oligonucleotide amplification reaction. Here we present two distinct types of this biphasic reaction chemistry and propose dominant reaction pathways for each type based on thermodynamic arguments. These reactions create an endogenous switch-like output that reacts to approximately 1pM oligonucleotide trigger. The chemistry is isothermal and can be adapted to respond to a broad range of input target molecules such as proteins, genomic bacterial DNA, viral DNA, and microRNA. This rapid DNA amplification reaction could potentially impact a variety of disciplines such as synthetic biology, biosensors, DNA computing, and clinical diagnostics.

scholarly journals A mathematical model for a biphasic DNA amplification reaction

2019 ◽  
Vol 16 (154) ◽  
pp. 20190143 ◽  
Author(s):  
Danielle Ciesielski ◽  
Burcu Özay ◽  
Stephanie McCalla ◽  
Tomas Gedeon
Keyword(s):  
Mathematical Model ◽  
Reaction Mechanisms ◽  
Dna Amplification ◽  
High Gain ◽  
Nucleic Acid Amplification ◽  
Dna Yield ◽  
Dna Oligonucleotides ◽  
Amplification Method ◽  
Analyte Detection ◽  
Amplification Reaction

Isothermal DNA amplification reactions are a prevalent tool with many applications, ranging from analyte detection to DNA circuits. Exponential amplification reaction (EXPAR) is a popular isothermal DNA amplification method that exponentially amplifies short DNA oligonucleotides. A recent modification of this technique using an energetically stable looped template with palindromic binding regions demonstrated unexpected biphasic amplification and much higher DNA yield than EXPAR. This ultrasensitive DNA amplification reaction (UDAR) shows high-gain, switch-like DNA output from low concentrations of DNA input. Here we present the first mathematical model of UDAR based on four reaction mechanisms and show the model can reproduce the experimentally observed biphasic behaviour. Furthermore, we show that three of these mechanisms are necessary to reproduce biphasic experimental results. The reaction mechanisms are (i) positively cooperative multistep binding spurred by two trigger binding sites on the template; (ii) gradual template deactivation; (iii) recycling of deactivated templates into active templates; and (iv) polymerase sequestration. These mechanisms can potentially illuminate the behaviour of EXPAR as well as other nucleic acid amplification reactions.

scholarly journals 661 PB 075 POLYMORPHIC DNA PROFILES AID IN DISTINGUISHING PETUNIA SPECIES

HortScience ◽  
1994 ◽  
Vol 29 (5) ◽  
pp. 527e-527
Author(s):  
Teresa A. Cerny ◽  
Terri W. Starman
Keyword(s):  
Wild Species ◽  
Morphological Traits ◽  
Gc Content ◽  
Dna Amplification ◽  
Seed Source ◽  
Banding Patterns ◽  
Dna Template ◽  
Dna Profiles ◽  
O 10 ◽  
Amplification Reaction

Our overall objective was to use DNA Amplification Fingerprinting (DAF) to determine the relationships between Petunia × hybrida and four wild petunia species,P. axillaris, P. inflata, P. parodii, and P. violacea. This research was to optimize the DAF amplification reaction for petunias, check for variability in the fingerprints among different seedlings of the same species and screen primers to be used for Identifying polymorphisms between cultivars of P. × hybrida end the four wild species. Optimization of the DAF procedure was accomplished by varying concentrations of DNA template (O - 10 ng), MgCl2(0 - 10 mM), and primers (0 - 30 μM). Optimum concentrations were found to be 1.0 ng DNA template and 2.0 mM MgCl2. Clearly resolved banding patterns were produced using primer concentrations from 3.0 μM to 30 μM. When separate seedlings of each wild species from the same seed source were fingerprinted, profiles were consistent. Seeds from other sources are presently being collected to investigate variation between sources. Twenty-five heptamer and octomer primers varying in GC content were screened and ten produced clear banding patterns for the Petunia species. These primers have produced polymorphic profiles between the pink-flowering species and the white-flowering species. Several primers have shown distinct polymorphisms between P. axillaris and P. parodii, the two white-flowering species, which have very similar morphological traits. Similarities in the banding patterns have been found between P. × hybrida and these wild species.

scholarly journals Cyclic Olefin Copolymer Microfluidic Devices for Forensic Applications

Biosensors ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 85 ◽  
Author(s):  
Brigitte Bruijns ◽  
Andrea Veciana ◽  
Roald Tiggelaar ◽  
Han Gardeniers
Keyword(s):  
Chemical Resistance ◽  
Multiple Displacement Amplification ◽  
Visible Region ◽  
Dna Amplification ◽  
Microfluidic Devices ◽  
Cyclic Olefin Copolymer ◽  
Color Test ◽  
Cyclic Olefin ◽  
On Chip ◽  
Amplification Reaction

Microfluidic devices offer important benefits for forensic applications, in particular for fast tests at a crime scene. A large portion of forensic applications require microfluidic chip material to show compatibility with biochemical reactions (such as amplification reactions), and to have high transparency in the visible region and high chemical resistance. Also, preferably, manufacturing should be simple. The characteristic properties of cyclic olefin copolymer (COC) fulfills these requirements and offers new opportunities for the development of new forensic tests. In this work, the versatility of COC as material for lab-on-a-chip (LOC) systems in forensic applications has been explored by realizing two proof-of-principle devices. Chemical resistance and optical transparency were investigated for the development of an on-chip presumptive color test to indicate the presence of an illicit substance through applying absorption spectroscopy. Furthermore, the compatibility of COC with a DNA amplification reaction was verified by performing an on-chip multiple displacement amplification (MDA) reaction.

scholarly journals Species diversity and geographic distribution of Gymnotus (Pisces: Gymnotiformes) by nuclear (GGAC)n microsatellite analysis

2000 ◽  
Vol 23 (4) ◽  
pp. 803-807 ◽  
Author(s):  
F.M.C. Fernandes-Matioli ◽  
S.R. Matioli ◽  
L.F. Almeida-Toledo
Keyword(s):  
Species Diversity ◽  
Geographic Distribution ◽  
Nuclear Genome ◽  
Dna Amplification ◽  
River Basins ◽  
Microsatellite Analysis ◽  
Species Specific ◽  
Single Primer ◽  
Amplification Reaction ◽  
Simple Sequence

Patterns of amplified DNA fragments flanked by (GGAC)n microsatellites, obtained by single primer amplification reaction (SPAR), from 198 Gymnotus specimens (Pisces: Gymnotiformes) sampled from 8 southeastern Brazilian river basins were analyzed. The species studied were Gymnotus carapo, G. pantherinus, G. inaequilabiatus, and G. sylvius. The indirectly obtained patterns reflected the distribution of simple sequence repeats in the nuclear genome of the specimens. Species-specific patterns of DNA amplification were found and were useful for the analysis of the geographic distribution of Gymnotus species. Monomorphic patterns were found in G. carapo, G. pantherinus, and G. inaequilabiatus. Three polymorphic patterns were found in G. sylvius populations. The SPAR technique could be a useful molecular tool in conservation programs involving communities of neotropical freshwater fish.

scholarly journals Komparasi Metode Isolasi DNA Patogen Antraknosa dan Bulai untuk Deteksi PCR

2016 ◽  
Vol 12 (4) ◽  
pp. 124
Author(s):  
Ade Syahputra ◽  
Kikin Hamzah Mutaqin ◽  
Tri Asmira Damayanti
Keyword(s):  
Pure Culture ◽  
Dna Isolation ◽  
Total Yield ◽  
Dna Amplification ◽  
Colletotrichum Acutatum ◽  
Isolation Method ◽  
Peronosclerospora Sorghi ◽  
Dna Purity ◽  
Dna Template ◽  
Commercial Kit

Polymerase chain reaction (PCR) is an important tool for detection, identification and monitoring of quarantine pests in Indonesia. DNA isolation method from target organism is an important step to provide adequate DNA template for performing PCR. Objective of the research was to compare conventional, commercial kit, FTA-card and its modification methods of DNA isolation to be used in PCR detection for Colletotrichum acutatum and Peronosclerospora sorghi from chili and maize, respectively. DNA obtained from various isolation methods were measured using UV-vis nanodrop-spectrophotometry.  DNA amplification was performed using DNA concentration of 15 ng µL-1 from each isolation method with gradual primer concentrations of 0.4; 0.6; 0.8; and 1.0 mM. The highest concentration of DNA was achieved with conventional methods for C. acutatum from pure culture and P. sorghi from maize leaf. Best DNA purity was obtained from isolation method using commercial kit for C. acutatum from infected fruit (1.94) and from conventional method for C. acutatum from pure culture (1.91). The highest total yield of isolated DNA was achieved by modified FTA-card for C. acutatum from pure culture. In general DNA amplification using various primer concentration gave positive results although DNA bands intensity was varied from faint to very bright.  Furthermore PCR optimization using the best primer concentration from previous reaction showed that all DNA templates resulted in thick and bright DNA bands.

scholarly journals First characterization of a biphasic, switch-like DNA amplification

The Analyst ◽  
2018 ◽  
Vol 143 (8) ◽  
pp. 1820-1828 ◽  
Author(s):  
Burcu Özay ◽  
Cara M. Robertus ◽  
Jackson L. Negri ◽  
Stephanie E. McCalla
Keyword(s):  
Dna Amplification ◽  
High Gain

An isothermal, high-gain DNA amplification chemistry with biphasic and switch-like properties.

Evolution of the Nucleoli During Oogenesis in Xenopus Laevis Studied by Electron Microscopy

1972 ◽  
Vol 10 (2) ◽  
pp. 339-367 ◽  
Author(s):  
P. VAN GANSEN ◽  
A. SCHRAM
Keyword(s):  
Xenopus Laevis ◽  
De Novo ◽  
Dna Amplification ◽  
Cellular Growth ◽  
Phase Iii ◽  
Second Phase ◽  
Nucleolar Structure ◽  
Third Phase ◽  
Electron Microscopes ◽  
Germinal Cells

Xenopus laevis tadpoles and toads were killed at several ages. The structure of the nuclei of the germinal cells has been observed by light and electron microscopes. We distinguish 11 successive stages in nucleolar structure: (1) a single, essentially granular nucleolus in the oogonium (10 µm diameter), (2) a reticulated nucleolus in the leptotene oocyte, (3) fragmentation of this nucleolus into a few smaller nucleoli, (4) multiple tiny nucleoli appearing in the cap of the pachytene oocyte, (5) enrichment in the fibrillar constituent of these intra-cap nucleoli, (6) grouped spherical nucleoli, with well segregated granular and fibrillar constituents, as the disintegration of the cap is going on (diplotene A oocyte, 30 µm diameter), (7) dispersion of those nucleoli in the nuclear sap (diplotene B oocyte, 50 µm diameter), (8) formation of long, ribboned nucleoli with multiple DNA-rich spots (diplotene C oocyte, 100 µm diameter), (9) fragmentation of the nucleolar ribbons into multiple spherical nucleoli with excentric fibrillar core and granular cortex (diplotene D oocyte, 150 µm diameter), (10) multiple purely fibrillar nucleoli (diplotene E oocyte, between 150 and 400 µm diameter), and (11) multiple classical nucleoli with concentric fibrillar core and granular cortex (diplotene F oocyte, between 400 and 1000 µm diameter). The multiplication of the nucleoli in Xenopus laevis may occur successively (a) by the fragmentation of the single oogonium nucleolus at the leptotene stage, (b) by de novo formation of nucleolar bodies inside the cap at the pachytene stage, and (c) by the growth of those nucleoli lying free in the nucleolar sap at the early diplotene stage. They evolve into nucleolar ribbons which later on fragment into spherical bodies. Four successive phases during the growth of an oocyte can be distinguished with respect to the ribosomal system. (I) The first phase is characterized by the nucleolar DNA amplification. (II) During the second phase, the multiplication of the nucleoli is going on. Ribosomes are present in the cytoplasm and the rate of cellular growth is very high. (III) During the third phase, the synthesis of rRNAs seem to be repressed while the synthesis of heterogenous small RNAs is going on. Ribosomes are no longer visible in the cytoplasm. The nucleoli are purely fibrillar. The rate of cell growth is lower than in the preceding phase. (IV) During the fourth ( = Duryee lampbrush stages 3-6), or vitellogenic phase, rRNAs are actively synthesized and numerous ribosomes appear in the cytoplasm. The nucleoli have the classical structure and the rate of growth is about the same as during phase III.

Inhibition of in vitro enzymatic DNA amplification reaction by ultra-violet light irradiation

1993 ◽  
Vol 7 (3) ◽  
pp. 217-219 ◽  
Author(s):  
Chia C. Pao ◽  
Jyu Jen Hor ◽  
Pei Ling Tsai ◽  
Ming Yow Horng
Keyword(s):  
Ultra Violet ◽  
Dna Amplification ◽  
Light Irradiation ◽  
Ultra Violet Light ◽  
Violet Light ◽  
Amplification Reaction

Genomic DNA amplification by the multiple displacement amplification (MDA) method

2009 ◽  
Vol 37 (2) ◽  
pp. 450-453 ◽  
Author(s):  
Roger S. Lasken
Keyword(s):  
Environmental Samples ◽  
Genomic Dna ◽  
Whole Genome Amplification ◽  
Multiple Displacement Amplification ◽  
Genomic Analysis ◽  
Dna Amplification ◽  
Genomic Sequencing ◽  
Extensive Coverage ◽  
Multiple Displacement Amplification Reaction ◽  
Amplification Reaction

Large amounts of DNA are frequently required for use in detection assays and genomic analysis. The limited availability of DNA can be a critical obstacle to meeting research and clinical needs. DNA amplification methods are often required to generate sufficient material from small specimens or environmental samples with low DNA content. The MDA (multiple displacement amplification) reaction is increasingly the method of choice for many applications because of its extensive coverage of the genome, the generation of extremely long DNA products compared with older whole genome amplification methods and the high DNA yields, even from exceedingly low amounts of starting material. Remarkably, MDA enables genomic sequencing even from single microbial cells. Some of the uses of MDA and its strengths and limitations will be discussed.

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