| 1. |
- Dias, Rita, et al.
(författare)
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Electrophoretic properties of complexes between DNA and the cationic surfactant cetyltrimethylammonium bromide
- 2005
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 26:15, s. 2908-2917
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Tidskriftsartikel (refereegranskat)abstract
- We use agarose gel electrophoresis to characterize how the monovalent catioinic surfactant cetyltrimethylammonium bromide (CTAB) compacts double-stranded DNA, which is detected as a reduction in electrophoretic DNA velocity. The velocity reaches a plateau at a ratio R = 1.8 of CTAB to DNA-phosphate charges, i.e., above the neutralization point, and the complexes retain a net negative charge at least up to R = 200. Condensation experiments on a mixture of two DNA sizes show that the complexes formed contain only one condensed DNA molecule each. These CTAB-DNA globules were further characterized by time-resolved measurements of their velocity inside the gel, which showed that CTAB does not dissociate during the migration but possibly upon entry into the gel. Using the Ogston-model for electrophoresis of spherical particles, the measured in-gel velocity of the globule is quantitatively consistent with CTAB having two opposite effects, reduction of both the electrophoretic charge and DNA coll size. In the case of CTAB the two effects nearly cancel, which can explain why opposite velocity shifts (globule faster than uncomplexed DNA) have been observed with some catioinic condensation agents. Dissociation of the complexes by addition of anionic surfactants was also studied. The DNA release from the globule was complete at a mixing ratio between anionic and cationic surfactants equal to 1, in agreement with equilibrium studies. Circular DNA retained its supercoiling, and this demonstrates a lack of DNA nicking in the compaction-release cycle which is important in DNA transfection and purification applications.
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| 2. |
- Bosaeus, Niklas, 1982, et al.
(författare)
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Force-induced melting of DNA-evidence for peeling and internal melting from force spectra on short synthetic duplex sequences
- 2014
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 42:12, s. 8083-8091
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Tidskriftsartikel (refereegranskat)abstract
- Overstretching of DNA occurs at about 60-70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.
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| 3. |
- Bosaeus, Niklas, 1982, et al.
(författare)
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Tension induces a base-paired overstretched DNA conformation
- 2012
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:38, s. 15179-15184
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Tidskriftsartikel (refereegranskat)abstract
- Mixed-sequence DNA molecules undergo mechanical overstretching by approximately 70% at 60-70 pN. Since its initial discovery 15 y ago, a debate has arisen as to whether the molecule adopts a new form [Cluzel P, et al. (1996) Science 271: 792-794; Smith SB, Cui Y, Bustamante C (1996) Science 271: 795-799], or simply denatures under tension [van Mameren J, et al. (2009) Proc Natl Acad Sci USA 106: 18231-18236]. Here, we resolve this controversy by using optical tweezers to extend small 60-64 bp single DNA duplex molecules whose base content can be designed at will. We show that when AT content is high (70%), a force-induced denaturation of the DNA helix ensues at 62 pN that is accompanied by an extension of the molecule of approximately 70%. By contrast, GC-rich sequences (60% GC) are found to undergo a reversible overstretching transition into a distinct form that is characterized by a 51% extension and that remains base-paired. For the first time, results proving the existence of a stretched basepaired form of DNA can be presented. The extension observed in the reversible transition coincides with that produced on DNA by binding of bacterial RecA and human Rad51, pointing to its possible relevance in homologous recombination.
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| 4. |
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| 5. |
- Carlsson, Nils, 1978, et al.
(författare)
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Bicontinuous cubic phase of monoolein and water as medium for electrophoresis of both membrane-bound probes and DNA
- 2006
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 22:9, s. 4408-4414
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Tidskriftsartikel (refereegranskat)abstract
- Porous hydrogels such as agarose are commonly used to analyze DNA and water-soluble proteins by electrophoresis. However, the hydrophilic environment of these gels is not suitable for separation of important amphiphilic molecules such as native membrane proteins. We show that an amphiphilic liquid crystal of the lipid monoolein and water can be used as a medium for electrophoresis of amphiphilic molecules. In fact, both membrane-bound fluorescent probes and water-soluble oligonucleotides can migrate through the same bicontinuous cubic crystal because both the lipid membrane and the aqueous phase are continuous. Both types of analytes exhibit a field-independent electrophoretic mobility, which suggests that the lipid crystal structure is not perturbed by their migration. Diffusion studies with four membrane probes indicate that membrane-bound analytes experience a friction in the cubic phase that increases with increasing size of the hydrophilic headgroup, while the size of the membrane-anchoring part has comparatively small effect on the retardation.
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| 6. |
- Carlsson, Nils, 1978, et al.
(författare)
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Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides
- 2005
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Ingår i: JOURNAL OF PHYSICAL CHEMISTRY B. ; 109:39, s. 18628-18636
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Tidskriftsartikel (refereegranskat)abstract
- We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6.
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| 7. |
- Carlsson, Nils, 1978, et al.
(författare)
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DNA hosted and aligned in aqueous interstitia of a lamellar liquid crystal - a membrane-biomacromolecule interaction model system
- 2013
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 9:33, s. 7951-7959
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Tidskriftsartikel (refereegranskat)abstract
- We report that DNA molecules can be intercalated and macroscopically oriented in the aqueous interstitia of a lyotropic lamellar liquid crystal. Using UV-vis linear dichroism and fluorescence spectroscopy we show that double-stranded oligonucleotides (25 base pairs) in the water-octanoate-decanol system remain base-paired in the B conformation and are confined in two dimensions, with the helix axis preferentially parallel to the lipid bilayer surfaces but free to rotate within this plane. The degree of helix confinement and the corresponding 2-D orientation can be improved by decreasing the thickness of the water interstitia via the fraction of water in the ternary mixture. Not surprisingly, the corresponding single-stranded oligonucleotides are not aligned, with their persistence length being short in comparison to the lamellar interstitium thickness. We propose this as a model system for studying interactions of DNA-ligand complexes near a lipid bilayer membrane which we demonstrate by using dye probes that are either covalently attached to one end of the oligonucleotide or reversibly bound by intercalation between the base pairs. Three cationic dyes, all strongly bound by intercalation to DNA when free in solution, are found to not bind to DNA but to prefer the membrane surface. The covalently attached Cy5 also binds to the bilayer while Cy3 tends to end-stack to the oligonucleotide duplex. The orientation of Cy5 parallel to the membrane indicates that electrostatic surface binding predominates over insertion into the hydrophobic interior of the membrane. Anionic and zwitterionic dyes (FAM and ROX) are found to remain randomly oriented in the water between the lipid bilayer surfaces. This journal is © The Royal Society of Chemistry.
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| 8. |
- Carlsson, Nils, 1978, et al.
(författare)
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Enzymes Immobilized in Mesoporous Silica: a Physical-Chemical Perspective
- 2014
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Ingår i: Advances in Colloid and Interface Science. - : Elsevier BV. - 0001-8686. ; 205, s. 339-360
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Forskningsöversikt (refereegranskat)abstract
- Mesoporous materials as support for immobilized enzymes have been explored extensively during the last two decades, primarily not only for biocatalysis applications, but also for biosensing, biofuels and enzyme-controlled drug delivery. The activity of the immobilized enzymes inside the pores is often different compared to that of the free enzymes, and an important challenge is to understand how the immobilization affects the enzymes in order to design immobilization conditions that lead to optimal enzyme activity. This review summarizes methods that can be used to understand how material properties can be linked to changes in enzyme activity. Real-time monitoring of the immobilization process and techniques that demonstrate that the enzymes are located inside the pores is discussed by contrasting them to the common practice of indirectly measuring the depletion of the protein concentration or enzyme activity in the surrounding bulk phase. We propose that pore filling (pore volume fraction occupied by proteins) is the best standard for comparing the amount of immobilized enzymes at themolecular level, and present equations to calculate pore filling from the more commonly reported immobilized mass. Methods to detect changes in enzyme structure upon immobilization and to study the microenvironment inside the pores are discussed in detail. Combining the knowledge generated from these methodologies should aid in rationally designing biocatalyst based on enzymes immobilized in mesoporous materials.
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| 9. |
- Carlsson, Nils, 1978, et al.
(författare)
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Quantification of protein concentration by the Bradford method in the presence of pharmaceutical polymers
- 2011
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 411:1, s. 116-121
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Tidskriftsartikel (refereegranskat)abstract
- We investigated how the Bradford assay for measurements of protein released from a drug formulation may be affected by a concomitant release of a pharmaceutical polymer used to formulate the protein delivery device. The main result is that polymer-caused perturbations of the Coomassie dye absorbance at the Bradford monitoring wavelength (595 nm) can be identified and corrected by recording absorptionspectra in the region of 350–850 mm. The pharmaceutical polymers Carbopol and chitosan illustrate two potential types of perturbations in the Bradford assay, whereas the third polymer, hydroxypropylmethylcellulose (HPMC), acts as a nonperturbing control. Carbopol increases the apparent absorbance at 595 nm because the polymer aggregates at the low pH of the Bradford protocol, causing a turbidity contribution that can be corrected quantitatively at 595 nm by measuring the sample absorbance at 850 nm outside the dye absorption band. Chitosan is a cationic polymer under Bradford conditions and interacts directly with the anionic Coomassie dye and perturbs its absorption spectrum, including 595 nm. In this case, the Bradford method remains useful if the polymer concentration is known but should be used with caution in release studies where the polymer concentration may vary and needs to be measured independently.
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| 10. |
- Carlsson, Nils, 1978, et al.
(författare)
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Spectroscopic characterization of Coomassie blue and its binding to amyloid fibrils
- 2012
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 420:1, s. 33-40
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Tidskriftsartikel (refereegranskat)abstract
- Coomassie brilliant blue G-250 (CB) is the dye used frequently in the Bradford assay for proteinconcentration determination. In this study, we investigated how the solvent polarity and viscosity affectthe CB absorption and fluorescence spectra and apply this understanding to investigate the binding of CBto lysozyme and insulin in the native and amyloid fibril states. Coomassie blue binds both to the nativeprotein and to amyloid fibrils but gives distinctly different spectral responses. The absorption andfluorescence spectra of CB indicate that binding sites in the fibrils are less polar and hold the CB dye morerigidly than in the native forms. The spectral comparison of CB bound to the two different fibrils showedthat the binding sites are different, and this was most likely due to differences in secondary structure asmonitored by circular dichroism. Finally, linear dichroism was used to show that the fibril-bound CB isoriented preferentially parallel to the insulin amyloid fibril axis.
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| 11. |
- Cole, K. D., et al.
(författare)
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Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels
- 2006
-
Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 27:22, s. 4396-4407
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Tidskriftsartikel (refereegranskat)abstract
- The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.
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| 12. |
- Dehkordi, Maryarn Nejat, et al.
(författare)
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Interaction of DNA with water soluble complex of Nickle and formation of DNA cross-links
- 2018
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Ingår i: Chemico-Biological Interactions. - : Elsevier BV. - 1872-7786 .- 0009-2797. ; 282, s. 55-62
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Tidskriftsartikel (refereegranskat)abstract
- Interaction of double stranded DNA with bulky and hydrophobic Salen type Schiff base complex: [N, N′ Bis [3- tert-butyl-5-[triphenyl-phosphonium – methyl] - salicylidene] 1,2 ethylene-diamine nickel(III) acetate (refer to Ni Salen complex) was extensively investigated using the spectroscopic techniques and gel electrophoresis. Absorption titration experiment showed the hypochromic effect and the significant red shift of the complex absorption. In competition experiments with ethidium bromide (EB), Ni Salen complex exhibited non-competitive binding at high concentrations. UV-vis absorption and fluorescence emission data agreed on a binding constant of (1.64 ± 0.01) μM −1 , thereby showing the strong interaction of the complex with DNA; also, a binding site size of 2.33 ± 0.01 base pairs per complex was achieved. Thermal denaturation experiment showed that T m of calf thymus-DNA was increased by approximately 10 °C at a molar ratio of the dye/base of 0.2. The CD spectra of DNA exhibited an increase in both positive and negative peaks without any shift in the position of bands upon addition of the complex. The amplitude of the LD spectra of DNA was decreased in the presence of the complex. Reduced l inear dichroism (LD red ) revealed that the transition moment of complex was parallel to the DNA helix axis. Gel electrophoresis experiments confirmed that Ni Salen complex had no nuclease/DNA cleaving activity; also, DNA-DNA cross links were formed at high concentrations of complex, leading to the aggregation of DNA.
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| 13. |
- Eriksson, Maja, et al.
(författare)
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Binding of Intercalating and Groove-Binding Cyanine Dyes to Bacteriophage T5
- 2007
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Ingår i: The Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 111:5, s. 1139-1148
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between four related cyanine dyes and bacteriophage T5 is investigated with fluorescence and absorption spectroscopy. The dyes, which differ in size, charge, and mode of DNA-binding, penetrate the capsid and bind the DNA inside. The rate of association decreases progressively with increasing dye size, from a few minutes for YO to more than 50 h for YOYO (at 37 °C). The relative affinity for the phage DNA is a factor of about 0.2 lower than for the same T5-DNA when free in solution. Comparison of groove-bound BOXTO-PRO and intercalating YO-PRO shows that the reduced affinity is not due to DNA extension but perhaps influenced by competition with other cationic DNA-binding agents inside the capsid. Although, the extent of dye binding to the phages decreases with increasing external ionic strength, the affinity relative to free DNA increases, which indicates a comparatively weak screening of electrostatic interactions inside the phage. The rate of binding increases with increasing ionic strength, reflecting an increase in effective pore size of the capsid as electrostatic interactions are screened and/or a faster diffusion of the dye through the DNA matrix inside the capsid as the DNA affinity is reduced. A combination of electron microscopy, light scattering, and linear dichroism show that the phages are intact after YO-PRO binding, whereas a small degree of capsid rupture cannot be excluded with BOXTO-PRO.
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| 14. |
- Eriksson, Maja, 1975, et al.
(författare)
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Comparing mono- and divalent DNA groove binding cyanine dyes--Binding geometries, dissociation rates, and fluorescence properties
- 2006
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Ingår i: Biophysical Chemistry. - : Elsevier BV. - 1873-4200 .- 0301-4622. ; 122:3, s. 195-205
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Tidskriftsartikel (refereegranskat)abstract
- The unsymmetrical cyanine dyes BOXTO-PRO and BOXTO-MEE were derived from the DNA groove binder BOXTO, by adding a positively charged or a non-ionic hydrophilic tail to BOXTO, respectively. The main objective was to obtain more efficient DNA probes, for instance in electrophoresis and microscopy, by slowing down the dissociation of BOXTO from DNA. The interactions with mixed sequence DNA was studied with fluorescence and absorbance spectroscopy, stopped-flow dissociation and gel electrophoresis. Both the derivatives are groove bound as BOXTO, and have similar fluorescence properties when bound to mixed sequence DNA in free solution. BOXTO-PRO exhibits a slower dissociation than BOXTO from DNA, whereas the dissociation rate for BOXTO-MEE is faster and, unexpectedly independent of the ionic strength. During gel electrophoresis both BOXTO-PRO and BOXTO-MEE exhibit a faster dissociation rate than BOXTO. Still, BOXTO-PRO seems to be a good alternative as DNA probe, especially for applications in free solution where the dissociation is slower than for the corresponding intercalator TOPRO-1.
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| 15. |
- Eriksson, M., et al.
(författare)
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Time-resolved electrophoretic analysis of mobility shifts for dissociating DNA ligands
- 2005
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Ingår i: Electrophoresis. - : Wiley. - 0173-0835 .- 1522-2683. ; 26:3, s. 524-532
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Tidskriftsartikel (refereegranskat)abstract
- Intercalative binding of ligands to DNA can be demonstrated by helix unwinding, monitored by gel electrophoresis of supercoiled DNA, as electrophoretic mobility is sensitive to the topological DNA state. However, we show that an apparent lack of unwinding in an electrophoretic assay could be due to dissociation of the (intercalated) ligand during the analysis, rather than evidence for a nonintercalative mode of binding to DNA. Repetitive scanning during the electrophoresis ensures that release of the ligand during electrophoresis does not affect the measured degree of unwinding, based on the electrophoretic velocity being determined as a function of time. We use this assay to establish intercalation as a mode of binding to DNA for the cyanine dyes YO, YO-PRO as well as two enantiomeric forms of the ruthenium complexes [(phen)2 Ru(tatpp)Ru(phen)2]4+, and to support groove-binding for the new unsymmetrical cyanine dyes BOXTO and BOXTO-PRO. Groove-binding could be concluded from a lack of unwinding, because we could rule out that it is caused by release of the dye during the electrophoresis. The gel electrophoresis has the advantage over hydrodynamic techniques that much smaller sample amounts are required, and our time-resolved approach can be employed in all mobility-shift assays when applied to dissociating complexes.
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| 16. |
- Hanczyc, Piotr, 1985, et al.
(författare)
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DNA in a polyvinyl alcohol matrix and interactions with three intercalating cyanine dyes
- 2011
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 115:42, s. 12192-12201
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Tidskriftsartikel (refereegranskat)abstract
- We investigate how DNA interacts with drugs in humid polyvinyl alcohol (PVA) films by using a homologous set of cyanine dyes (YO(+), YO-PRO(2+), and YOYO(4+)) known to intercalate into DNA with increasing affinity with increasing charge. UV-vis spectroscopy shows that the PVA matrix destabilizes all three DNA-dye complexes compared to aqueous solution but to a lesser degree as the dye charge increases. The monovalent YO is fully dissociated from DNA within minutes, whereas the dissociation of the divalent YO-PRO takes about one hour and occurs by a two-step mechanism. The tetravalent homodimer YOYO is even less affected by the PVA environment and remains intercalated in the B-form DNA also in the PVA films. The reduced stability of the DNA-dye complexes is discussed in terms of steric and dielectric properties of the PVA matrix. After being kept in dry PVA films for 48 h the DNA-YOYO complexes can be reformed reversibly by rehumidifying the films for 30 min. The ability to store aligned and confined DNA intercalated with ligand complexes may be useful in studies on structural properties of nucleic acids.
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| 17. |
- Hanczyc, Piotr, 1985, et al.
(författare)
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Short Oligonucleotides Aligned in Stretched Humid Matrix – Secondary DNA structure in Poly(Vinyl Alcohol) Environment
- 2012
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 1520-5827 .- 0743-7463. ; 28:16, s. 6662-6669
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Tidskriftsartikel (refereegranskat)abstract
- We report that short, synthetic, double- as well as single-stranded DNA can be aligned in stretched humid poly(vinyl alcohol) (PVA) matrix, and the secondary structure (nucleobase orientation) can be characterized with linear dichroism (LD) spectroscopy. Oligonucleotides of lengths varying between 10 (3.4 nm) and 60 bases (20.4 nm) were investigated with respect to structural properties in the gel-like polymer environment. The DNA conformation as a function of relative humidity reveals a strong dependence of helical structure of DNA on PVA hydration level, results of relevance for nanotechnical studies of DNA-based supramolecular systems. Also, the PVA gel could provide possibilities to test models for nucleic acid interactions and distribution in cell contexts, including structural stability of genetic material in the cell and PVA-packaging for gene delivery. A method by which duplex oligonucleotides, with sequences designed to provide specific binding sites, become amenable to polarized-light spectroscopy opens up new possibilities for studying structure in DNA complexes with small adduct molecules as well as proteins.
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| 18. |
- Johansson, Johan, 1980, et al.
(författare)
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Covalent functionalization of carbon nanotube forests grown in situ on a metal-silicon chip
- 2012
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Ingår i: Proceedings of SPIE - The International Society for Optical Engineering. - : SPIE. - 0277-786X .- 1996-756X. - 9780819490018 ; 8344
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Konferensbidrag (refereegranskat)abstract
- We report on the successful covalent functionalization of carbon nanotube (CNT) forests, in situ grown on a silicon chip with thin metal contact film as the buffer layer between the CNT forests and the substrate. The CNT forests were successfully functionalized with active amine and azide groups, which can be used for further chemical reactions. The morphology of the CNT forests was maintained after the functionalization. We thus provide a promising foundation for a miniaturized biosensor arrays system that can be easily integrated with Complementary Metal-Oxide Semiconductor (CMOS) technology.
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| 19. |
- Jonsson, Mats, 1939, et al.
(författare)
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Orientation of DNA during gel electrophoresis studied with linear dichroism spectroscopy
- 1988
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Ingår i: Biopolymers. - : Wiley. - 0006-3525 .- 1097-0282. ; 27:3, s. 381-414
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Tidskriftsartikel (refereegranskat)abstract
- A method for in situ study of orientation of DNA during gel electrophoresis has been developed. Linear dichroism spectra measured by this phase-modulation technique can sensitively and selectively detect orientation of DNA during electrophoretic migration in gel. [Measurement of “electrophoretic orientation” was first reported in 1985 by B. Åkerman, M. Jonsson, and B. Nordén (1985) (J. Chem. Soc. Chem. Commun. 422–423)]. Restriction fragments of duplex DNA of lengths in the ranges of 300–2319 base pairs (bp) and 4361–23130 bp have been studied in 5% polyacrylamide and 1% agarose gels, respectively. The fragments become preferentially oriented with the DNA helix axis parallel to the migration direction. In agarose the orientation is found to increase sigmoidally, and in polyacrylamide, linearly, with the electric field strength, within the field ranges accessible to measurement (0–40 and 5–40 V/cm, respectively). In both types of gels a considerable increase in orientation with length of DNA was observed. Compared to dipole orientation in electric fields, the electrophoretic orientation is high: orientation factor S = 0.027 in agarose for 23130 bp at 10 V/cm and S = 0.004 in polyacrylamide for 2319 bp at 10 V/cm. In addition to orientation of DNA, the electrophoresis also leads to orientation effects in the gel structure owing to Joule heating. In agarose there is also an effect that is associated with the migrating DNA zones and that produces different orientations of the gel at the front and rear parts of a zone. Evidence is presented that this effect is due to a DNA-induced electroosmotic flow causing a contraction of the gel in the front of the zone and an expansion in the rear. The experimental results on DNA orientation are compared with the reptation theories for gel electrophoresis. The theory of Lumpkin et al. [O. J. Lumpkin, P. Dejardin, and B. H. Zimm (1985) Biopolymers24, 1573–1593] predicts no orientation length dependence, but it does predict a shape of the field dependence that resembles the shape observed in agarose. The theory of Slater and Noolandi [G. W. Slater and J. Noolandi (1986) Biopolymers25, 431–454] predicts an orientational length dependence that is an order of magnitude less than the experimental one, and a field dependence that agrees neither with the sigmoidal shape observed in agarose nor with the linear dependence in polyacrylamide.
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