摘要:

AbstractData‐adaptive algorithms are presented for separating overlapping signatures of heterozygotic allele pairs in electrophoresis data. Application is demonstrated for human microsatellite CA‐repeat polymorphisms in LiCor 4000 and ABI 373 data. The algorithms allow overlapping alleles to be called correctly in almost every case where a trained observer could do so, and provide a fast automated objective alternative to human reading of the gels. The algorithm also supplies an indication of confidence level which can be used to flag marginal cases for verification by eye, or as input to later stages of statistical analy

ISSN:0173-0835    

DOI:10.1002/elps.1150180102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes persist within electrophoresis gels for much longer than expected on the basis of their free‐solution lifetimes. Volume exclusion, direct interaction with gel matrices and the reduction of water activity by the gel have been proposed as mechanisms enhancing the stability of complexes during electrophoresis. We have used the well‐characterized interaction of theE. colicyclic AMP receptor protein (CAP) with lactose promoter DNA to test these proposals. We found that the activity of water within polyacrylamide gels differs little from that of the buffer in which they were cast and that the dependence of the dissociation rate constant on water activity is too small for osmotic stabilization to contribute significantly to the lifetimes of CAP‐DNA complexes. In addition, we found that a cross‐linked gel matrix is not required for the stabilization of CAP‐DNA complexes, that comparable stabilization is produced by three dissimilar polymers (linear polyacrylamide, dextran and polyethylene glycol), and that these polymers stabilize complexes more effectively than equivalent weight concentrations of their cognate monomers. While these results challenge the notion that direct interaction with the gel matrix contributes to the stability of protein‐DNA complexes, they are all features expected of excluded volume

ISSN:0173-0835    

DOI:10.1002/elps.1150180103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractAntiparallel complementary strands of DNA contribute symmetrically to the helical structure and its stability; rough conformational differences between complementary strands would interfere with this symmetric contribution and the stability. To detect conformational differences between complementary strands, we challenged the complementary strands to different “pore size” of acrylamide gels and measured differences in migration during electrophoretic mobility analysis. Changes in migration differences revealed a significant structural bias between the complementary strands of the dinucleotide repeat deoxy‐(TG

ISSN:0173-0835    

DOI:10.1002/elps.1150180104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe feasibility of using nanofabricated arrays as electrophoretic chambers for DNA sequencing is investigated. A specific array design, consisting of rows of closely spaced posts, separated by longer open spaces, is proposed. Molecules driven through the array by an electric field get hooked over obstacles at successive rows and their progress through the device is delayed as a consequence. The dependence of the delay time on molecular size is derived. Numerical evaluation indicates that a device of modest dimensions, operating at high fields, can rapidly resolve oligonucleotides containing several hundred bases.

ISSN:0173-0835    

DOI:10.1002/elps.1150180105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractIn a previous paper (Yinet al., Electrophoresis1996,17, 1143–1150), an automated method for matrix determination in four‐dye fluorescence‐based DNA sequencing was presented. As a continuation of that work, we have developed an alternative method to estimate the matrix from raw sequence data. The method uses an iterative clustering technique to associate each 4 × 1 data vector with one column of the desired filter matrix, using Kullback's I‐divergence as a distance measure. The method requires less preprocessing of the data and less computation than the approach described by Yinet al.(Electrophoresis1996,17, 1143–1150). An example demonstrating applicability of the proposed method to Applied Biosystems sequencer dat

ISSN:0173-0835    

DOI:10.1002/elps.1150180106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA model of electrophoretic mobility of small charged particles (for example short DNA fragments) in media with curved channels is proposed. The medium is represented by a dense material embedding the curved channels accessible to the charged particles. The steady flow method is used to obtain an analytical expression of the particles' electrophoretic mobility as a function of the channel's shape. The analogy between the statistical properties of the channels and polymer chains (free‐jointed and with the persistent mobility mechanism) is use

ISSN:0173-0835    

DOI:10.1002/elps.1150180107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe dependence of DNA mobility anomalies on gel pore size has been studied in polyacrylamide gels with a wide variety of compositions, using molecular weight ladders containing multiple copies of normal (12B) and anomalously slowly migrating (12A) 147‐base pair restriction fragments from plasmid pBR322 as the migrating probe molecules. If the gel pore size is increased by decreasing the total acrylamide concentration (%T) at constant cross‐linker ratio (%C), the usual method of increasing gel pore size, the mobility anomalies decrease with increasing gel pore radius as though the 12A multimers were retarded by a sieving mechanism. However, the decrease in the mobility anomalies is independent of whether the apparent gel pore radius is larger or smaller than the DNA radius of gyration, suggesting that gel pore size is not the controlling variable. If the acrylamide concentration is held constant and the gel pore size is increased by decreasing %C at constant %T, the mobility anomalies of the largest 12A multimers (6 mers and higher) decrease with increasing gel pore radius, because of sieving effects, until the effective gel pore radius becomes approximately equal to the DNA radius of gyration, after which the mobility anomalies level off and become independent of gel pore size. The mobility anomalies exhibited by 5‐mers and smaller multimers of fragment 12A are independent of gel pore radius in all gels with constant %T. Similar results are observed with a molecular weight ladder containing phased A‐tracts from the kinetoplast bending locus. Since the anomalous electrophoretic mobilities depend primarily on the total acrylamide concentration in the gel, and not on the apparent gel pore radius, increases in the magnitude of the mobility anomalies with increasing gel concentration (and decreasing gel pore radius) cannot be taken as evidence for DNA cu

ISSN:0173-0835    

DOI:10.1002/elps.1150180108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractRenal cell carcinoma is the most common neoplastic disease of the adult kidney and occurs in its sporadic and hereditary form. Approximately 57% of all renal carcinomas of the clear cell type analyzed revealed a mutation in the von Hippel‐Lindau disease (VHL) gene. In the present work, temperature gradient gel electrophoresis (TGGE) is presented as a rapid and precise polymerase chain reaction (PCR)‐employing methodology for the detection of mutations in the VHL gene. The theoretical efficiency of TGGE to detect mutations in every base pair of the gene was calculated. According to computer analysis, at least 92% of all known mutations in the VHL gene are detectable. This calculated figure appears to be in agreement with the experimental results. Primary difficulties in analyzing exon 1 of the VHL gene were overcome by the employment of psoralen‐cross‐linked PCR fragments. In addition, TGGE analysis was used in screening for possible mutations in thirteen renal carcinoma samples. With this protocol TGGE is successfully added to the array of methods for the screening of VHL mu

ISSN:0173-0835    

DOI:10.1002/elps.1150180109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractA strategy is described for detecting mitochondrial (mt) DNA variation which permits rapid and straightforward screening for forensic purposes. The method is based on the selection of fragments with adequate length for performing single strand conformation polymorphism (SSCP) analysis selecting a set of restriction enzymes (RE) which yield fragments with prefixed lengths. After digestion of mtDNA by the appropriate enzyme or set of enzymes, SSCP analysis is performed in a semiautomatic electrophoretic system using a silver staining detection method. The conformational changes due to single mutations were therefore found not to change the electrophoretic protocol but to change the relative position of the mutations within the fragment. The discrimination power of this method is estimated to be 90% when two restriction enzymes (MspI andHinfl) are used, but it is considerably higher when other enzymes are added.

ISSN:0173-0835    

DOI:10.1002/elps.1150180110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY

摘要:

AbstractThe pore size of agarose gel in water at different concentrations was directly measured using atomic force microscopy (AFM). The experiment was specially designed to work under aqueous conditions and allows direct observation of the “unperturbed” gel without invasive treatment. The pore sizeaas a function of gel concentrationCshows a power law dependencea ˜ C−γ, where γ lies between the prediction of the Ogston model for a random array of straight chains, 0.5, and the value predicted by De Gennes for a network of flexible chains, 0.75. We confirm that gels present a wide pore size distribution and show that it narrows as the concentration i

ISSN:0173-0835    

DOI:10.1002/elps.1150180111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1997

数据来源: WILEY