Improved Single-Strand DNA Sizing Accuracy in Capillary Electrophoresis

Abstract

Interpolation algorithms can be developed to size unknown single-stranded (ss) DNA fragments based on their electrophoretic mobilities, when they are compared with the mobilities of standard fragments of known sizes; however, sequence-specific anomalous electrophoretic migration can affect the accuracy and precision of the called sizes of the fragments. We used the anomalous migration of ssDNA fragments to optimize denaturation conditions for capillary electrophoresis. The capillary electrophoretic system uses a refillable polymer that both coats the capillary wall to suppress electro-osmotic flow and acts as the sieving matrix. The addition of 8 M urea to the polymer solution, as in slab gel electrophoresis, is insufficient to fully denature some anomalously migrating ssDNA fragments in this capillary electrophoresis system. The sizing accuracy of these fragments is significantly improved by the addition of 2-pyrrolidinone, or increased capillary temperature (60 degrees C). the effect of these two denaturing strategies is additive, and the best accuracy and precision in sizing results are obtained with a combination of chemical and thermal denaturation.

Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices

“…(i) Discrepancies between in silico and experimental T-RF sizes, i.e., 1.6 Ϯ 1.5 units in this study, may reduce specific inference of particular T-RFs in complex profiles (30,52). Migration discrepancies for different T-RF sequences with the same length led to differences between in silico and experimental T-RFLP profiles (Fig.…”

Spinocerebellar ataxias: microsatellite and allele frequency in unaffected and affected individuals

“…In CE, amplicons were found to migrate faster than predicted, and the results show a lower number of repeats in one or two peaks [29][30][31] . Factors contributing to these discrepancies could be the different sieving effects of cross-linked PAGE compared with the linear polymer used in CE, the electroosmotic effect, the extent of denaturation or differences in secondary structure between amplicons and standard markers 32 . Considering the previous studies regarding differences between PAGE with CE, we did not compare these two methodologies.…”

Segregation distortion of wild-type alleles at the Machado-Joseph disease locus: a study in normal families from the Azores islands (Portugal)

“…Amplicon length was calculated by comparison with the GeneScan 500-TAMRA size standard using the GeneScan Analysis 3.1.2 software. Size standard fragments 250 bp and 340 bp were not considered for size estimation purposes due to their anomalous migration (Rosenblum et al 1997). A size correction formula [(CE product size (bp) -198)/3 9 1.0184 + 0.7062; adapted from Dorschner et al 2002] was applied to allow the comparison with data from previous works, where manual polyacrylamide gel electrophoresis (PAGE) assay was used instead of CE.…”