摘要:

AbstractIt has previously been shown that zones of higher electric field form close to the loading end of the gel during denaturing polyacrylamide gel electrophoresis. Here we show that the field can reach up to three times its normal mean value a few cm in front of the loading wells when 44.5 mMTris‐44.5 mMboric acid‐1 mMEDTA is used as the gel buffer. We also demonstrate that this electric field gradient is mostly due to the difference in ion transference numbers at the gel/buffer interface caused by the high viscosity of the urea solution contained in the gel. This field gradient leads to increased band widths and forces us to redefine both the electrophoretic mobility and the mean field intensity. We discuss some methods that can be used to minimize the effects of this gradi

ISSN:0173-0835    

DOI:10.1002/elps.1150190503

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

年代:1998

数据来源: WILEY

摘要:

AbstractThe electrophoresis of small DNA fragments has been measured in dilute agarose and polyacrylamide gels cast and run in Tris‐acetate‐EDTA (TAE) and Tris‐borate‐EDTA (TBE) buffers. Ferguson plots were constructed to extrapolate the mobilities to zero gel concentration and estimate the free solution mobility of DNA. In polyacrylamide gels, in both TAE and TBE buffers, the extrapolated mobilities at zero gel concentration increased gradually with decreasing DNA molecular weight, went through a maximum at ∼ 60 bp, and then decreased again. The increase in the extrapolated mobilities with decreasing molecular weight observed for DNA fragments ⩾ 60 bp can be attributed to transient interactions between the migrating DNA molecules and the polyacrylamide gel fibers. If such interactions are eliminated by extrapolating the mobilities to both zero gel concentration and zero DNA molecular weight, the apparent free solution mobility of DNA is found to be 3.1 × 10−4cm2V−1s−1in TAE buffer and 4.2 × 10−4cm2V−1s−1in TBE buffer at 20°C, reasonably close to the actual free solution mobilities measured in the same two buffers by capillary electrophoresis (N. C. Stellwagenet al.,Biopolymers1997,42, 687–703). The significantly larger electrophoretic mobility observed in TBE buffer is most likely due to the formation of nonspecific, highly charged deoxyriboseborate complexes in this buffer medium. For DNA molecules ⩽ 60 bp in size, the decrease in the extrapolated mobilities with decreasing molecular weight parallels the decrease in their free solution mobilities observed by capillary electrophoresis. In agarose gels, the extrapolated mobilities of small DNA molecules at zero gel concentration appear to be independent of molecular weight. The apparent free solution mobilities are found to be (3.0 ± 0.1) × 10−4cm2V−1s−1in TAE buffer and (3.2 ± 0.1) × 10−4cm2V−1s−1in TBE buffer. The very similar mobilities observed in the two buffer media suggest that the borate ions in TBE buffer primarily form complexes with the galactose residues in the agarose gel fibers, rather than with the migrating DNA molecules, because of mass action effects. The formation of borate‐agarose complexes, increasing the net negative charge of the agarose gel fibers, appears to be responsible for the markedly increased electroendosmotic flow observed in agarose gels cast and run in TBE buffer (N

ISSN:0173-0835    

DOI:10.1002/elps.1150190504

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

年代:1998

数据来源: WILEY

摘要:

AbstractAn enhancement of hybridization labeling signals is demonstrated in Southern blotted DNAs, fractionated by voltage gradient gel electrophoresis. This enhancement is due to a reduced thickness of each single nucleic acid band in the gel as a consequence of the gradient effect, corresponding to an increased concentration of DNA per unit area.

ISSN:0173-0835    

DOI:10.1002/elps.1150190505

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

年代:1998

数据来源: WILEY

摘要:

AbstractA simple procedure for the isolation of genes as DNA fragment lengths is described. By using a commercial continuous elution protein electrophoresis apparatus and incorporating an agarose matrix, preparative scale amounts (300 μg) of DNA can be purified by fragment lengths from a mixture of genomic fragment lengths with high recovery yields. Fractions corresponding to unique fragment length ranges are screened for individual genes by dot‐blot analysis. Using this technique, we have isolated two genes:PGK1, a single copy housekeeping gene; andp53(exons 3–11), a tumor suppressor gene, whose DNA fragment lengths elute at 4 and 7.5 kbp, respectively, from a single preparative run. As an example of the utility of the technique, we applied it to improving the sensitivity of the ligation‐mediated polymerase chain reaction (LMPCR) — a nucleic acid amplification technique used for the detection and mapping of DNA damage along genes. By eliminating excess nontargeted genomic DNA, the agarose matrix continuous elution electrophoresis (CEE) procedure provided a 24‐fold increase in signal strength attributable to base damage caused by exposing DNA to reactive oxygen species. Genomic DNA fragment length purification by agarose matrix CEE should also prove useful in other research areas requiring gene isolation, such as genomics and molecul

ISSN:0173-0835    

DOI:10.1002/elps.1150190506

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

年代:1998

数据来源: WILEY

摘要:

AbstractCutaneous T cell lymphomas (CTCL) can be differentiated from benign inflammatory dermatoses by the demonstration of clonal T cells in skin biopsy. As a marker of the T cell clonality, the rearrangement of the T cell receptor (TCR) genes is amplified by polymerase chain reaction (PCR) and subsequently analyzed by several electrophoresis techniques. Since the validity of this approach depends substantially on the separating capacity of the applied electrophoresis technique, we investigated in the present study the lower detection limit and the sensitivity of heteroduplex‐loaded polyacrylamide gel electrophoresis on MDE® (mutation detection enhancement) gels (HD‐MDE PAGE), of heteroduplex‐loaded temperature gradient gel electrophoresis (HD‐TGGE) and fragment analysis (FA) on sequencing gels. Genomic DNA from formalin‐fixed, paraffin‐embedded skin biopsies of 53 CTCL specimens and 27 samples of benign dermatoses was analyzed by TCRyPCR followed by electrophoretic separation. Clonality was detected by HD‐MDE PAGE in 22, by HD‐TGGE in 34, and by FA in 33 of the 53 CTCL cases. Additionally, FA revealed an oligoclonal fragment profile in seven CTCL specimens. In the 27 samples from benign dermatoses, HD‐MDE PAGE and HD‐TGGE showed the expected polyclonal pattern in 26, and FA in 25 specimens. HD‐TGGE and FA detected a clonal pattern down to a dilution of 103monoclonal cells in 106peripheral blood mononuclear cells (PBMC), while HD‐MDE PAGE revealed a detection limit of 104monoclonal cells in 106PBMC. In conclusion, HD‐TGGE and FA possess a higher sensitivity and lower detection limit than HD‐MDE PAGE. Therefore, both former techniques are useful tools for the routine diagnostic procedure. With regard to time an

ISSN:0173-0835    

DOI:10.1002/elps.1150190507

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

年代:1998

数据来源: WILEY

摘要:

AbstractGenotyping of the alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci is important in alcohol studies. We describe a method for simulataneous genotyping of ADH2 and ALDH2 based on amplified product length polymorphism (APLP) analysis. Two polymerase chain reaction (PCR) fragments for ADH2 (57 bp, 53 bp) and two for ALDH2 (78 bp, 73 bp) are simultaneously amplified. Nine banding patterns reflecting the genotypes of the ADH2 and ALDH2 loci are clearly and unambiguously distinguished. The APLP method seems to be particularly suited for large‐scale population studies of ADH2 and ALDH2 loci because it is simple and rapi

ISSN:0173-0835    

DOI:10.1002/elps.1150190508

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

年代:1998

数据来源: WILEY

摘要:

AbstractThe AT‐rich variable number of tandem repeat (VNTR) marker at the 3′ end of the collagen type IIα1 (COL2A1) gene has been shown to have a complex structure with extensive sequence variations among repeat units. We analyzed this VNTR polymorphism in a large population of 972 Caucasian individuals with a genotyping procedure involving heteroduplex analysis of PCR products using polyacrylamide gel electrophoresis. Seven size alleles were identified, combining to 19 different homoduplex genotypes (heterozygosity = 0.64). These could be further dissected by analysis of heteroduplexes into 85 different heteroduplex genotypes (heterozygosity = 0.84). By systematically heteroduplexing homozygous and heterozygous individualsin vitro, characteristic heteroduplex doublet bands were generated of known allelic composition. A comparison of these doublets with heteroduplex patterns observed in the population allowed us to identify 29 alleles. The degree of correspondence with a different COL2A1 VNTR genotyping system, based on size separation of single‐strand VNTR alleles [l], was investigated by the analysis of samples typed with both methods, including samples from reference CEPH pedigrees. This revealed improved genetic resolution by the heteroduplex method including discrimination of frequent alleles considered identical by the single‐strand analysis. Our findings demonstrate heteroduplex analysis of the COL2A1 VNTR to be a robust and highly informative genetic marker system. The documented increased genetic variability has important implications in forensic and paternity testing, as well as in linkage and association studies relating genetic variation at this locus to disease e

ISSN:0173-0835    

DOI:10.1002/elps.1150190509

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

年代:1998

数据来源: WILEY

摘要:

AbstractSingle‐strand conformation polymorphism (SSCP) analysis combined with automated laser fluorescence detection is proposed as a comprehensive, rapid and sensitive method for screening sequence variation of the human beta‐actin‐related pseudogene (HUMACTBP2). Eleven sequenced alleles representing each type of known sequence variant of HUMACTBP2 locus were studied. Allelic variants of the same size but different sequence structures are easily resolved on the basis of their secondary conformation. Fifty ACTBP2 amplification products previously typed on a denaturing gel were repeatedly examined to determine the utility of SSCP analysis in terms of ease of interpretation and reproduction capabilities of the conformational patterns. Eleven sequenced ACTBP2 allelic variants were used as external conformation standards in polymerase chain reaction (PCR)‐SSCP subtyping. This enabled identification of polymorphism in a particular length variant and therefore consistent discrimination between heterozygous samples appeared identical on denaturing gels. Of five “homozygous” samples, one was shown to be heterozygous for two distinct alleles of the same size but different sequences. Thus, the method provides a unique possibility for detecting false homozygotes. The technique complements both denaturing gel electrophoresis and DNA sequencing in studies on the overall variability of the A

ISSN:0173-0835    

DOI:10.1002/elps.1150190510

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

年代:1998

数据来源: WILEY

摘要:

AbstractThis study evaluated the use of a commercially available DNA intercalating agent (Resolver Gold™) in agarose gels for the direct detection of sequence variation in ribosomal DNA (rDNA). This agent binds preferentially to AT sequence motifs in DNA. Regions of nuclear rDNA, known to provide genetic markers for the identification of species of parasitic ascarid nematodes (order Ascaridida), were amplified by polymerase chain reaction (PCR) and subjected to electrophoresis in standard agarose gelsversusgels supplemented with Resolver Gold. Individual taxa examined could not be distinguished reliably based on the size of their amplicons in standard agarose gels, whereas they could be readily delineated based on mobility using Resolver Gold‐supplemented gels. The latter was achieved because of differences (approximately 0.1–8.2%) in the AT content of the fragments among different taxa, which were associated with significant interspecific differences (approximately 11–39%) in the rDNA sequences employed. There was a tendency for fragments with higher AT content to migrate slower in supplemented agarose gels compared with those of lower AT content. The results indicate the usefulness of this electrophoretic approach to rapidly screen for sequence variability within or among PCR‐amplified rDNA fragments of similar sizes but differing AT contents. Although evaluated on rDNA of parasites, the approach has potential to be applied to a range of genes of different groups of infectious

ISSN:0173-0835    

DOI:10.1002/elps.1150190511

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

年代:1998

数据来源: WILEY

摘要:

AbstractHydrophobic proteins in the cell wall of the opportunistic fungal pathogenCandida albicansare involved in adhesion of this organism to host tissue and thus play a role in its pathogenicity. The hydrophobic nature of these proteins results in their loss during purification due to adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic proteins in sufficient quantity for sequencing or biochemical analysis. We describe a system that combines preparative isoelectric focusing with continuous elution preparative electrophoresis. The system provides a two‐dimensional protein separation while maintaining protein solubility and minimizing protein loss due to adsorption. In addition, we have added an in‐line transfer of electrophoretic fractions directly to polyvinylidene difluoride (PVDF) membranes, which further reduces both exposure to apparatus surfaces and purification t

ISSN:0173-0835    

DOI:10.1002/elps.1150190512

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

年代:1998

数据来源: WILEY