Detection of DNA curvature by transverse pore gradient gel electrophoresis on PhastSystem gels.

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of "Ferguson curves" with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800-900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.     

Information on DNA conformation derived from transverse pore gradient gel electrophoresis in conjunction with an advanced data analysis applied to capillary electrophoresis in polymer media.

Abnormally slow migration of DNA is conventionally viewed as being due to an abnormal conformation relative to "linear" standards. The evidence for this rests on a few instances where nonlinear DNA structures have been established by independent methods and yield low mobilities relative to standards. Transverse pore gradient gel electrophoresis of authentically bent kinetoplast DNA and of an upstream activator sequence (UAS) of an E. coli operon promoter shows in addition that curves of migration distance vs. gel concentration ("Ferguson curves") of such abnormally conformed DNA differ from those of "linear" standards. Since Ferguson curves are interpretable with regard to molecular size in concordance with a mathematical model (Ogston model), transverse pore gradient gel electrophoresis provides a simple means of correlating abnormally slow migration of DNA with molecular size. In addition, transverse pore gradient gel electrophoresis is able to distinguish between DNA banding which exhibits a steeper dependence on gel concentration than "linear" standards from one which shows the same dependence. The former appears characteristic of circularly bent DNA and gives rise to a substantial retardation, the latter of bending across a knot or kink in the DNA chain associated with a relatively minor retardation relative to standards. Circularly bent restriction fragments formed from kinetoplast DNA retain the characteristic intersecting Ferguson curves on the transverse pore gradient gel. Another authentically "abnormal" DNA structure recognizable on transverse pore gradient gels is supercoiled DNA derived from the reaction of topoisomerase with a plasmid. Different lengths of supercoiled sequences give rise to parallel Ferguson curves clearly intersecting with those of linear standards.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free mobility determination by electrophoresis in polyacrylamide containing agarose at a nonrestrictive concentration

In the determination of the free mobility, related to the surface net charge, by quantitative gel electrophoresis, the previous arbitrary extrapolation of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0% T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5% agarose to polyacrylamide at the various low concentrations in application to a DNA fragment 155 bp in size (Orbán, L. et al., in preparation). The present study applies that approach to several proteins and DNA fragments smaller than 1300 bp, using 0.4% agarose in polyacrylamide gels of varying concentration. The intercepts of the plots with the mobility axis provide experimental data by which the free mobility in polyacrylamide gel electrophoresis can be estimated for molecules not significantly retarded in their migration at the agarose concentration admixed to polyacrylamide. Across the gel concentration range below 3% T, in the presence of agarose, the Ferguson plots of proteins and DNA fragments are convex. It was shown by mass spectrometry that this convex curvature of the plots in the mixed polymer is not significantly due to low polymerization efficiency in the concentration range of liquid polyacrylamide (below 3%T).     

The electric field dependence of DNA mobilities in agarose gels: a reinvestigation

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

样品稀释对毛细管电泳分离检测DNA片段的影响

通过理论推导和实验验证表明;适当稀释DNA样品溶液,采用流体力学进样或电动进样都不会较大地减低峰高,而DNA片段毛细管电泳的分离效率和分离度还能有所提高。采用稀释样品的方法可提高DNA样品的使用效率。采用羟乙基纤维素无胶筛分介质分离了DNA片段。用激光诱导荧光（氩离子激光器,488nm）电荷耦合器件检测。用低浓度的筛分介质（0．4％）分离了分子质量较大的ADNA－HindⅢ全部8个片段（12bp～23130bP）。用高浓度的筛分介质（1．6％）分离分子质量较小的pBR322－HaeⅢ22个片段（18bp～587bp）。     

Estimation of polyacrylamide gel pore size from Ferguson plots of normal and anomalously migrating DNA fragments. I. Gels containing 3% N,N'-methylenebisacrylamide

The mobilities of normal and anomalously migrating DNA fragments were determined in polyacrylamide gels of different acrylamide concentrations, polymerized with 3% N,N'-methylenebisacrylamide as the crosslinker. The DNA samples were a commercially available 123-bp ladder and two molecular weight ladders containing multiple copies of two 147-base pair (bp) restriction fragments, obtained from the MspI digestion of plasmid pBR322. One of the 147 bp fragments is known to migrate anomalously slowly in polyacrylamide gels. Ferguson plots were constructed for all multimer ladders, using both absolute mobilities and relative mobilities with respect to the smallest DNA molecule in each data set. If the retardation coefficients were calculated from the relative mobilities, and the rms radius of gyration was used as the measure of DNA size, the Ogston equations were obeyed and the gel fiber parameters could be calculated. The effective pore sizes of the gels were estimated from the gel concentration at which the mobility of a given DNA molecule was reduced to one-half its mobility at zero gel concentration. The estimated pore radii ranged from approximately 130 nm for 3.5% gels to approximately 70 nm for 10.5% gels. These values are much larger than the pore sizes previously determined for the polyacrylamide matrix.     

Hydrogen-bonding interactions in the active sites of cytochrome P450cam and its site-directed mutants.

Resonance Raman spectroscopy is applied to the cyanide adducts of cytochrome P450cam and its T252A and D251N site-directed mutants, both in their substrate-free and camphor-bound forms, to probe active-site heme structure and, in particular, interactions of the FeCN fragment with potential active-site H-bond donors. In contrast to the ferrous CO and ferric NO adducts, which form only essentially linear (slightly distorted) FeXY fragments, the spectra of the ferric CN(-) adducts provide clear evidence the for the existence of an additional, rather highly bent, conformer; that is, the cyanide complexes form both linear and bent conformers in both the substrate-free and substrate-bound forms. Formation of this bent conformer is most reasonably attributed to the presence of off-axis H-bond donors, which induce distortion on the FeCN fragment but not the FeCO and FeNO fragments, which are poorer H-bond acceptors. For all three proteins, the substrate-free form exhibits a complex spectral pattern which arises because one of the modes associated with the FeCN fragment is coupled with two heme macrocycle deformation modes. Significantly, no evidence for such coupling is observed in the spectra of the camphor-bound forms. While various unknown factors may possibly give rise to selective activation of such coupling in the substrate-free derivative, given the known facts about the active-site architecture of this enzyme, a plausible explanation is that the bent conformer is oriented toward the water-filled substrate-binding site in the substrate-free form, but oppositely, toward the proposed proton delivery shuttle, in the substrate-bound form. Sensitivity of the FeCN modes to H(2)O/D(2)O exchange in the two camphor-bound mutants, which is apparently absent for the camphor-bound native protein, is most reasonably attributed to the known presence of extra water in the active sites of these mutants.     

DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

For first-line non-small-cell lung cancer(NSCLC) therapy, detecting mutation status of the epidermal growth factor receptor(EGFR) geneconstitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy. Now, the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded(FFPE) tissues. DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction(PCR) are two key steps for detecting EGFR gene mutation. We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene. Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100(pH=10.0) was performed by heating at 95℃ for 30 min. Meanwhile, a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison. DNA extracted products were used as template for amplifying the exons 18, 19, 20 and 21 of EGFR by PCR for different amplified fragments. Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250-500 base pairs(bp). However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp. The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit. For about 500 bp fragment, four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit. However, for about 200 bp fragment. This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR, further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.     

Improved DNA fragment length estimation in capillary electrophoresis

In CE the charged DNA strands are fractionated according to fragment lengths as they migrate through the capillary, since shorter DNA fragments pass through the sieving matrix faster. Multiplexed internal size standards are used to estimate the size of unknown DNA fragments. In the literature there are statements about migration abnormalities for the 250 and 340 bp fragments in the GeneScan-500 (GS500) internal size standards. Such anomalous migration of size standards could obviously introduce errors in the estimation of unknown fragments. Therefore, a number of analysis programs simply exclude some of these fragments. In the present work we first evaluate the effect of excluding each of the fragments in the internal size standards used in CE. Next, a method which is based on estimating the true values of the anomalous fragments is presented. The results obtained by the new method indicate a significant improvement compared to results obtained when one (or both) of the anomalous fragments in GS500 is (are) excluded or included when estimating the size of unknown DNA fragments. In the higher-molecular-weight region, the average error is reduced from 1.91 bp in ABI GeneMapper (excluding 250 bp) to 0.15 bp in the new method (using the estimated values for 250 and 340 bp). In the lower-molecular-weight region, excluding both fragments will improve the results by an average of 0.74 bp compared to ABI GeneMapper.     

Effect of total percent polyacrylamide in capillary gel electrophoresis for DNA sequencing of short fragments. A phenomenological model.

Polyacrylamide capillary gels were prepared with constant (5% C) cross-linker concentration and with total acrylamide concentration ranging from 2.5 to 6% T. At each acrylamide concentration, peak spacing was constant for DNA sequencing fragments ranging from 25 to 250 nucleotides in length. Peak spacing increased linearly with the total acrylamide concentration. The intercept of the retention time vs. fragment length plot was independent of % T. Ferguson plots were constructed for short DNA fragments; the polyacrylamide pore size falls in the 2.5 to 3.5 nm range for the gels studied. Theoretical plate count is independent of total acrylamide concentration; longitudinal diffusion, and not thermal gradients, limit the plate count. A phenomenological model is presented that predicts retention time, plate count, and resolution for sequencing fragments ranging in size from 25 to 250 bases and gels that range from 2.5 to 6% total acrylamide.     

线性聚丙烯酰胺凝胶毛细管电泳的迁移特性

使用线性聚丙烯酰胺作为筛分介质,对片段长度为80～584bp的标准DNA样品进行毛细管电泳,利用激光诱导荧光方法检测信号,荧光染料为溴化乙啶。改变电场强度100～375V/cm,得到的迁移率曲线与电场强度和DNA片段长度成复杂的函数关系,已有的经典理论模型:Ogston模型、Reptation无拉伸模型和Reptation拉伸模型都不能正确地描述实验观察到的迁移率随电场强度和DNA片段长度的变化情况。因此,提出一种修正的Ogston筛分理论,假定迁移的DNA分子在电场强度方向延展拉伸,如同小分子穿过凝胶筛孔。在该修正模型中,DNA的迁移率仅依赖于电场强度、筛分介质浓度和片段长度,很好地解释了实验现象。     

Unit cloning and amplification as novel and universal strategies for complex vector construction and small DNA fragment preparation

With a novel and universal strategy for the cloning of multiple DNA fragments, a complex synthetic vector (pVEC100), harboring the target DNA fragments in conventional 100‐bp DNA ladder, was constructed for efficient and large‐scale production of 100‐bp DNA marker through bacteria fermentation, plasmid extraction and restrictive digestion. Since the restrictive digestion of complex vectors yields insufficient small DNA fragments, an innovative PCR model was developed as an alternative. The PCR model comprised a specially designed template vector and a unit amplification model for producing groups of small DNA fragments. The unit amplification model improved the efficiency of the PCR protocol and made it more economical and easier for small DNA fragment amplification. The approach presented in this paper – a unit cloning model for constructing complex synthetic vectors combined with the modular design of unit amplification by PCR – is a powerful method for preparing small DNA fragments of DNA molecular weight standards.     

Curved DNA molecules migrate anomalously slowly in polyacrylamide gels even at zero gel concentration

The electrophoretic mobilities of curved and normal DNA molecules of the same size have been measured in polyacrylamide gels containing various acrylamide concentrations and cross-linker ratios. Ferguson plots were constructed to extrapolate the observed mobilities to zero gel concentration. The DNA samples were two 147-bp restriction fragments, called 12A and 12B, obtained from the MspI digestion of plasmid pBR322, and head-to-tail multimers of each fragment. Fragment 12A is stably curved and migrates anomalously slowly in polyacrylamide gels; fragment 12B has the conformation of normal DNA and migrates with normal electrophoretic mobilities. The extrapolated mobilities of the curved fragment 12A and its multimers at zero gel concentration are lower than the extrapolated mobilities of the normal fragment 12B and its multimers. The free solution mobility of the curved fragment 12A, measured by CE, is also lower than that of the normal fragment 12B. The combined results indicate that the extrapolated mobilities observed for curved DNA molecules at zero polyacrylamide gel concentration reflect the intrinsic differences in their free solution mobilities.     

Information on DNA conformation derived from the Ferguson plot of DNA fragments of up to 9 kb in size, using polyacrylamide gel electrophoresis in a discontinuous buffer system.

The Ferguson plot in polyacrylamide gel electrophoresis (PAGE)(15%CDATD, moving boundary electrophoresis buffer system operative at pH 8.9, 4 degrees C, 8 mA/cm2 of gel) of DNA fragments up to 9.4 kb in size was found to exhibit a linear segment at polyacrylamide concentrations starting at 3% T and undergoing a gradual transition into a concave segment at higher gel concentrations, confirming previous findings by Stellwagen. The larger the DNA, and the higher the gel concentration, the less extended the linear and the more extended the concave segment of the plot. The lowest % T of the linear range for DNA in polyacrylamide remains unknown since mobilities at nongelling concentrations below 3% T have not as yet been measured. As previously suggested, the transition from the linear to the concave segment corresponds to that from the randomly oriented DNA to the anisotropically stretched, "reptating" DNA. For a DNA of 9.4 kb in size, the end of the linear range of the Ferguson plot can be extended from 3.5 to 5% T when 15% DATD rather than 2.5% Bis is used to crosslink the polyacrylamide. Increasing the temperature of PAGE from 4 degrees C to 25 and 50 degrees C widens the linear segment progressively, indicating an increasingly random orientation with rising temperature. When current density is increased from 8 to 40 mA/cm2, the concave curvature of the Ferguson plot of DNA 1 to 9.4 kb in size decreases, suggesting a transition from a "reptating" to a randomly distributed molecule, due to increased Joule heat.(ABSTRACT TRUNCATED AT 250 WORDS)     

脱氧核糖核酸分子在高分子溶液3个不同浓度区间的电泳迁移行为

 借鉴高分子亚浓溶液线团收缩理论 ,研究了脱氧核糖核酸 (DNA)片段在高分子溶液全浓度区间的电泳迁移行为。结果表明 ,在高分子稀溶液、亚浓溶液和浓溶液 3个不同浓度区间 ,DNA的电泳迁移行为各不同 ,DNA片段的分离在这 3个浓度区间也存在差异。     

Estimation of polyacrylamide gel pore size from Ferguson plots of linear DNA fragments. II. Comparison of gels with different crosslinker concentrations, added agarose and added linear polyacrylamide.

The mobilities of various DNA fragments in two normally migrating molecular weight ladders were studied in polyacrylamide gels containing different concentrations of the crosslinker N,N'-methylenebisacrylamide (Bis). The acrylamide concentration ranged from 2.5-10.5%T (w/v); the Bis concentration ranged from 0.5-10%C (w/w), with respect to total acrylamide. Ferguson plots were constructed for each of the DNA fragments in gels of each composition. The Ferguson plots of the different multimers in each molecular weight ladder were nearly parallel in gels containing 0.5-3%C, converged close to a common intercept at zero gel concentration in gels containing 4%C, and crossed at approximately 1.5%T in gels containing 5 and 10%C. If the mobilities observed for the different DNA fragments at zero gel concentration were also extrapolated to zero DNA molecular weight, a common limiting mobility was observed in gels of all crosslinker concentrations. This limiting mobility was approximately equal to the free solution mobility of DNA. The effective pore radius of each gel was estimated from Ferguson plots based on relative mobilities, using the mobility of the smallest DNA fragment in each molecular weight ladder as the reference mobility. The calculated gel pore radii ranged from 142 nm to 19 nm, respectively, for gels containing 4.6%T, 1.5%C, and 10.5%T, 5 or 10%C. These pore radii are an order of magnitude larger than previously accepted values, but are consistent with scanning electron microscope measurements (Rüchel, R., et al., J. Chromatogr. 1978, 42, 77-90).(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of comparability in amplified fragment length polymorphism profiling using a simple model system

A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated. The reproducibility of the AFLP model was tested by submitting both "unknown" DNA template that had been restricted and ligated with AFLP linkers (R/L mixture) and corresponding primer pairs to nine laboratories participating in the study. Participants completed the final PCR step and then used either slab gel electrophoresis or CE to detect the AFLP fragments. The predicted fragments were identified by the majority of participants with size estimates consistently up to 3 base pair (bp) larger for slab gel electrophoresis than for CE. Shadow fragments, 3 bp larger than the predicted fragments, were often observed by study participants and organizers. The nine AFLP fragments exhibited relative intensities ranging from less than 3% to 22% and, apart from the two weakest fragments, with a % CV of 16 to 25. Fragments containing the highest guanine-cytosine (GC) content of 50-56% showed the greatest stability in the AFLP profiles.     

Disposable polyester-toner electrophoresis microchips for DNA analysis

Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a direct-printing process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300 V cm(-1). Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215,000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device.     

Enhanced π‐Backdonation from Gold(I): Isolation of Original Carbonyl and Carbene Complexes

The specific electronic properties of bent o‐carborane diphosphine gold(I) fragments were exploited to obtain the first classical carbonyl complex of gold [(DPCb)AuCO]⁺ (ν(CO)=2143 cm^?1) and the diphenylcarbene complex [(DPCb)Au(CPh₂)]⁺, which is stabilized by the gold fragment rather than the carbene substituents. These two complexes were characterized by spectroscopic and crystallographic means. The [(DPCb)Au]⁺ fragment plays a major role in their stability, as substantiated by DFT calculations. The bending induced by the diphosphine ligand substantially enhances π‐backdonation and thereby allows the isolation of carbonyl and carbene complexes featuring significant π‐bond character.