Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society最新文献

Factors other than molecular weight are known to affect DNA electrophoretic mobility. DNA methylation has been found to affect the curvature of DNA, causing anomalous mobility in polyacrylamide gels; the effect of methylation on the mobility of large DNA molecules in agarose gels was unknown. Chromosomal DNA from Mycoplasma capricolum, a wall-less prokaryote which has a low intrinsic methylation rate, was methylated in agarose blocks by SssI methylase, a de novo methylase with a CpG recognition sequence. (A surprising finding was that SssI methylase altered the structure of InCert, but not SeaKem Gold, agarose.) Restriction enzyme analysis was used to estimate the extent of CpG methylation. DNA methylation was found to have no effect on the electrophoretic mobility of full-length chromosomal DNA (1,120 kbp) in agarose gels. Therefore, methylation is not a source of error in PFGE-based size estimation for chromosomal DNA molecules less than 1.12 Mbp in agarose gels.

The analysis of crude polymerase chain reaction (PCR) products by capillary electrophoresis (CE) is often compromised by the presence of a high concentration of salt. Salt interferes with the electrokinetic injection and induces localized heating within the column, hence, PCR products must be desalted or cleaned-up prior to CE analysis. A variety of commercial clean-up systems are available that have been traditionally used to prepare PCR products for cloning, sequencing, and digestion with restriction enzymes. These systems were tested for their effectiveness in preparing PCR products for CE analysis and were evaluated based on CE resolution, salt removal, DNA recovery, processing time, and cost.

Using an isoelectric focusing gel containing agarose, urea, a separator and a narrow range ampholyte of pH 5-6, a system was designed to split the F13A*1 and F13A*2 alleles into the subtypes 1A, 1B, 2A and 2B. Four population groups (European-Americans, African-Americans, Mexican-Americans and Native-Americans) were data based. Subtyping F13A increased the information content significantly over the previous F13A typing system by doubling the number of alleles and increasing the number of phenotypes from three to ten. The new system has proved to be of value in parentage testing by increasing exclusionary power in cases of non-paternity and increasing the paternity index in non-exclusionary cases. Though there does not appear to be any significant variation among U.S. populations, published data on Japanese populations suggests that significant differences among populations may exist, leading to an anthropological usefulness as well.

TreviGel-500, a new polysaccharide matrix containing AgaCryl, commercially available as a powder for slab gel electrophoresis, is now being applied to the separation of DNA fragments in capillary electrophoresis. The capillary mode allows the use of one to two orders of magnitude lower mass fractions of matrix and approximately five to six orders of magnitude lower sample quantities than the slab gel electrophoresis counterpart for optimal separation of DNA fragments in the 100 to 2,000 base pair size range. In the capillary mode, this new separation matrix forms a semi-rigid gel that demonstrates enhanced selectivity for DNA fragments in the 1,000 to 7,000 base pair size range relative to alternative size-sieving polymer solutions. In addition, this matrix offers the advantages of lower toxicity than acrylamide. Comparisons are drawn between the use of this matrix in both slab gel electrophoresis and capillary electrophoresis for the separation of DNA fragments with respect to the mass fraction of the matrix in buffer, the buffer composition and sample loading or injection parameters.

Capillary electrophoresis (CE) is capable of rapid, high-resolution separations of DNA. The technique has been adopted for both ssDNA and dsDNA applications. In order to make CE more convenient and cost-effective, replaceable sieving buffers have recently been developed. For DNA sequencing, the most successful of these replaceable buffers have been carefully polymerized and purified viscous linear polyacrylamide solutions. However, the hazards of acrylamide are well documented, and the care required to prepare the appropriate molecular weight polymer make this approach less than ideal. We report use of a replaceable sieving buffer suitable for DNA sequencing by CE that is easy to prepare and uses commercially available, non-toxic hydroxyethylcellulose.

The use and development of discontinuous buffer systems for the separation of proteins and nucleic acids are reviewed, and the advantages over a continuous buffer system are presented. Emphasis is given to the more recent applications in DNA separations where discontinuous systems have aided in the resolution and mobility tailoring of DNA fragments. Various DNA size ranges can be separated in short time periods by simple choice of buffer components rather than gel alterations. Guidelines for choosing appropriate buffer systems are offered as well as the application of borate complexes to modify the mobility of DNA. This article reviews the historical development of discontinuous buffers in the electrophoresis of proteins and nucleic acids with emphasis on the characteristics and applicability to nucleic acid separations.

The 100-1,000 basepair size typical of PCR-amplified DNA fragments demands high resolution electrophoretic gels for the adequate characterization of small differences among samples. We have studied the behavior of a number of commercial sizing ladders in three classes of separation systems: polyacrylamides with discontinuous buffer, proprietary acrylamides with continuous buffer, and agarose-like materials with continuous buffer. None of the ladders examined perform adequately in any of these systems using vendor-supplied nominal ladder component basepair sizes. All ladders successfully typed D1S8O alleles after calibration with the allelic ladder (replacing the nominal size values with the least squares estimate of allele/matrix-specific apparent sizes). Some ladders and matrices are qualitatively better than others. No one ladder proved consistently better than others; a polyacrylamide gel with ribose modifier provided the most precise results in this study. Appropriately calibrated electrophoretic apparent sizes must be used for results to be validly exchanged among laboratories. Appropriate allelic ladders or a well defined subset of known alleles can serve as the calibration system.

Cell-free transcription and translation products from an ordered library of cDNA clones from the CEM human leukemic cell line were submitted to analysis using two-dimensional gel electrophoresis as a read out system. The matrix array of 24 x 16 x 12 wells contained in each of the positions lambda jacII phages from one plaque. Pools of clones along the three axes (24 x-pools, 16 y-pools and 12 z-pools) were established. Results obtained upon matching of 12 x-pools were scrutinized for estimating the frequency of cDNA molecular species in the library. The results obtained are interpreted in such a way that there are no discrete distributions of mRNA molecular species, but rather there is a continuous distribution of mRNA's covering a wide range of frequencies. The lowest frequency found was about 4.5 x 10(-4) and the highest 1.6 x 10(-2). About half of all clones can be found among these low frequency ones (each occurring 0.45 times among 1,000 clones).

We have analyzed an ordered library of 4,608 cDNA clones from the CEM human leukemic cell line. The aim was to facilitate gene retrieval, to enable immediate access to cDNA clones and to provide information on the protein expression of the individual clones in a 2D gel readout. The matrix array of 24 x 16 x 12, each position of which contained lambda jacII phage from one plaque, enabled us to establish pools of clones along the three axes (24 pools of complexity 192 cloned entities, 16 pools of complexity 288 and 12 pools of complexity 384). The total cDNA complexity is here reduced to such a level that spots which in more complex gels served as landmark spots are not present in each pool, and thus cannot serve as landmarks anymore. The image analysis of such gels and especially the matching of spots is not reliable under these circumstances. In order to achieve reliable matching, additional samples were created, such that pools were co-electrophoresed according to a special concatenation scheme; these samples then contained over-lapping elements (e.g., pools 1 + 2 + 3 and 3 + 4 + 5 have at least those spots in common, which originate from pool 3). This approach turned out to be feasible and we have completed the matching of one half of the ordered library. Already from the present stage of analysis we have obtained valuable information on the cDNA library and on the distribution of clones in this library.

We pursued the goal of obtaining global information on cDNA libraries from lymphocytes by combining data on the genes with data on the expressed proteins. Ordered libraries enabled us to establish the properties of the clones by direct retrieval of individual clones from the matrix (in our system a 24 x 16 x 12 array). We have picked-upon inspection of radiofluorographs obtained from the pools of the library--39 clones and performed a 2D gel analysis on the cell free expressed clonal products and have established also their partial nucleotide sequences. Details on two such clones are reported; clone CEM1001 is a new gene, while clone CEM1002 is a gene for human elongation factor 1-beta.