Room temperature method for increasing the rate of DNA reassociation by many thousandfold: the phenol emulsion reassociation technique

Kohne, David E.; Levison, Stuart A.; Byers, Michael

doi:10.1021/bi00643a026

Cited by 169 publications

(76 citation statements)

References 14 publications

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“…Previous data ruled out the possibility that small circular DNA consisted of repeats of a single short sequence (10), but no estimates of total sequence complexity were made due to the difficulty of obtaining sufficient material to generate high Cot values under standard aqueous conditions. By using the PERT (24), which greatly increases the rate of DNA/DNA reassociation, we were able to study the self-hybridization of small circular DNA with the small amount of material available. The PERT conditions used here increase the rate of hybridization for Escherichia coli DNA approximately 20,000-to 40,000-fold over that observed under standard aqueous (ECot) conditions.…”

Section: Resultsmentioning

confidence: 99%

Small circular DNA of Drosophila melanogaster: chromosomal homology and kinetic complexity.

Stanfield¹,

Lengyel²

1979

Proc. Natl. Acad. Sci. U.S.A.

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Nucleic acid reassociation techniques were used to determine the kinetic complexity of small circular DNA in cultured cells of Drosophila melanogaster. Two kinetic components are present. One of these constitutes 82% of the mass of the circular DNA and has a complexity of 1.8 X 104 nucleotide pairs; the other constitutes 18% of the mass and has a significantly higher but undefined sequence complexity. We have demonstrated that these circular molecules hybridize to middle repetitive chromosomal sequences by hybridization of in vitro-labeled circular DNA tracer with a vast excess of Drosophila chromosomal DNA. Thermal stability measurements indicate that base-pair mismatch between small circular DNA and middle repetitive chromosomal DNA does not exceed 2%. We discuss possible functions of these small circular DNAs in light of the above findings. Heterogeneous closed circular DNA is an intriguing and little understood class of molecules that has been described in a wide variety of eukaryotic organisms, including Neurospora, Euglena, trypanosomes, yeast, tobacco, Xenopus, and boar, and in cell culture lines from monkey, mouse, and human (1-9).We have investigated this class of molecules in embryos and cultured cells of Drosophila melanogaster (10) because of the unique genetic and cytological advantages that this organism offers for the study of structure and function of DNA sequences.In the cultured cells (Schoeider's line 2), small circular DNA is predominantly nuclear And exhibits a buoyant density in neutral CsCl indistinguishable from that of the main band nuclear DNA. The circular molecules range in size from approximately 340 to >7500 nucleotide pairs (Nt Pr) with an average size of 3300 Nt Pr. The size distribution and average circle size of small circular DNA in embryos and in a cloned subline of Schneider's line 2 cells differ significantly from that of Schneider's line 2. Both logarithmic and stationary phase cells contain a minimum of 3 to a maximum of 40 average-sized small circular DNA molecules per cell, constituting a maximum of 0.03% of the total cellular DNA (10).Although the function of heterogeneous circular DNA is unknown, it has been suggested that it might be involved in various genetic regulatory phenomena in Drosophila via integration into and excision from chromosomal DNA (11)(12)(13)(14)(15)(16)(17). Because an understanding of the function of these circular molecules depends upon the characterization of the sequences contained within them, we have determined the sequence complexity of Drosophila heterogeneous circular DNA (18) by a modification of the procedure of Stanfield and Helinski (10). After purification in three consecutive CsCl/ethidium bromide gradients, circular DNA was digested with RNase T1 (20 ,ug/ml), a-amylase (100 Atg/ml), and preboiled RNase A (20,tg/ml) in 15 mM NaCl/1.5 mM Na citrate, pH 7.2/0.02 M Na2 EDTA for 30 min at 370C. Sarkosyl was added to 1% and the DNA was recentrifuged on two consecutive analytical grade CsCl gradients to separate mitochondrial (...

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Section: Resultsmentioning

confidence: 99%

Small circular DNA of Drosophila melanogaster: chromosomal homology and kinetic complexity.

Stanfield¹,

Lengyel²

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…[45][46][47] In another, formation of an emulsion with the addition of phenol to water also increased the hybridization rate, which was attributed to interfacial adsorption and diffusion. 48,49 Because of its complexity, however, the mechanism of DNA hybridization is still under debate. 50 Herein, we report molecular beacon hybridization in nine different organic solvents up to 75% (v/v).…”

Section: Introductionmentioning

confidence: 99%

Fast Molecular Beacon Hybridization in Organic Solvents with Improved Target Specificity

Dave

Liu

2010

J. Phys. Chem. B

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DNA hybridization is of tremendous importance in biology, bionanotechnology, and biophysics.Molecular beacons are engineered DNA hairpins with a fluorophore and a quencher labeled on each of the two ends. A target DNA can open the hairpin to give an increased fluorescence signal. To date, the majority of molecular beacon detections have been performed only in aqueous buffers. We describe herein DNA detection in nine different organic solvents, methanol, ethanol, isopropanol, acetonitrile, formamide, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethylene glycol, and glycerol, varying each up to 75% (v/v). In comparison to detection in water, the detection in organic solvents showed several important features. First, the molecular beacon hybridizes to its target DNA in the presence of all the nine solvents up to a certain percentage. Second, the rate of this hybridization was significantly faster in most organic solvents compared to water. For example, in 56% ethanol the beacon showed a 70-fold rate enhancement. Third, the ability of the molecular beacon to discriminate single base mismatch is still maintained. Lastly, the DNA melting temperature in the organic solvents showed a solvent concentrationdependent decrease. This study suggests that molecular beacons can be used for applications where organic solvents must be involved or organic solvents can be intentionally added to improve the molecular beacon performance.

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“…In addition, the ability to renature complementary DNA strands in vitro (3)(4)(5) has become a widely used tool for the identification and analysis of specific nucleic acid sequences (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). For these and other reasons, considerable effort has gone towards understanding the mechanism of nucleic acid renaturation, as well as towards finding conditions where the rate of renaturation is enhanced (16)(17)(18)(19)(20)(21).…”

Section: Introductionmentioning

confidence: 99%

Rapid renaturation of complementary DNA strands mediated by cationic detergents: a role for high-probability binding domains in enhancing the kinetics of molecular assembly processes.

Pontius

Berg

1991

Proc. Natl. Acad. Sci. U.S.A.

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The rate of renaturation for complementary DNA strands can be enhanced >104-fold by the addition of simple cationic detergents, and the reaction is qualitatively and quantitatively very similar to that found with purified heterogeneous nuclear ribonucleoprotein Al protein. Under optimal conditions, renaturation rates are >2000-fold faster than reactions run in 1 M NaCI at 68°(. The reaction is second-order with respect to DNA concentration, and reaction rates approach or equal the rate with which complementary strands are expected to encounter each other in solution. Renaturation can even be observed well above the expected melting temperature of the duplex DNA, demonstrating that some cationic detergents have DNA double-helix-stab g properties. The reaction is also extremely rapid in the presence of up to a l06-fold excess of noncomplementary sequences, establishing that renaturation is specific and relatively independent of heterologous DNA. This finding also implies that up to several thousand potential target sequences can be sampled per strand per second. Such reagents may be useful for procedures that require rapid nucleic acid renaturation, and these results suggest ways to identify and design other compounds that increase the kinetics of association reactions. Moreover, this work provides further support for a model relating the existence of flexible, weakly interacting, repeating domains to their function in rapid molecular assembly processes in vitro and in vivo.The renaturation of nucleic acid strands is of fundamental importance in biological processes such as genetic recombination and repair (1,2). In addition, the ability to renature complementary DNA strands in vitro (3-5) has become a widely used tool for the identification and analysis of specific nucleic acid sequences (6-15). For these and other reasons, considerable effort has gone towards understanding the mechanism of nucleic acid renaturation, as well as towards finding conditions where the rate of renaturation is enhanced (16)(17)(18)(19)(20)(21).Recently, we and others demonstrated that the Al protein of heterogeneous nuclear ribonucleoprotein (hnRNP) can rapidly renature complementary strands of both DNA and RNA (refs. 22 and 23; X. Dong and S. Munroe, personal communication). This unanticipated property of Al was accounted for by its ability to bind to nucleic acid strands and present flexible, weakly interacting domains with a repeating structure (22). This model predicts that other molecules possessing flexible domains containing, for example, repeating hydrophobic residues, will also substantially increase the rate of nucleic acid renaturation when they are bound to complementary sequences. To test this hypothesis, as well as to identify readily available compounds that could be used to accelerate nucleic acid renaturation, we investigated the properties of two simple cationic detergents, dodecyl-and cetyltrimethylammonium bromide (DTAB and CTAB).DTAB and CTAB are variants of the quaternary amine tetramethylammonium bromide...

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Room temperature method for increasing the rate of DNA reassociation by many thousandfold: the phenol emulsion reassociation technique

Cited by 169 publications

References 14 publications

Small circular DNA of Drosophila melanogaster: chromosomal homology and kinetic complexity.

Small circular DNA of Drosophila melanogaster: chromosomal homology and kinetic complexity.

Fast Molecular Beacon Hybridization in Organic Solvents with Improved Target Specificity

Rapid renaturation of complementary DNA strands mediated by cationic detergents: a role for high-probability binding domains in enhancing the kinetics of molecular assembly processes.

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