Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis

Abstract

Denaturing CE (DCE) is a powerful tool for analysis of DNA variation. The development of commercial multi-CE instruments allows large-scale studies of DNA variation (many samples and many fragments). However, the cost of consumables like capillary arrays and sieving matrix might limit the use of DCE in such studies. Thus, we have tested 72 different in-house formulated sieving matrices' ability to suppress EOF and separate PCR-amplified alleles with the DCE variant, cycling temperature CE (CTCE). The data herein demonstrate that alleles can be baseline-separated by use of PVP and poly(N,N-dimethyl acrylamide) polymers at various percentages and pH. Allele separation by CTCE is matrix-independent and consequently applicable to any capillary instrument used for DNA separation. Formulation of sieving matrix for CTCE was done by dissolving appropriate amount of polymer powder into the running buffers. Allele separation was observed at different pH (7.5-8.5), concentrations and molecular size of the polymer, without compromising the separation and reproducibility. Finally, the cost reduction of homemade matrices is more than 1000-fold as compared to commercial sieving matrices.

Electrokinetic DNA transport in 20 nm‐high nanoslits: Evidence for movement through a wall‐adsorbed

“…In free solution the addition of long-chain 1 MDa PVP with a chain length of about 2.5 mm can entangle and retard DNA molecules and enable separation [45,46]. However, the 10 kDa PVP we use has an approximate chain length of only 25 nm [47].…”

Clinical Relevance of KRAS in Human Cancers

“…More than 60 methods described can be divided into sequencing methods [30–37], methods based on specific interaction with oligonucleotide, methods based on specific interaction with enzyme [38–40], and conformational methods [41–47]. While many specificity and/or sensitivity enhancement of methods were described as well [48–53], analytical validation, systematic comparison, and assessment of methods side by side is lacking.…”

Analysis of mutational spectra by denaturing capillary electrophoresis

“…It is clear that understanding of point mutagenesis during human development in multiple organs will require an increased capacity for mutational spectrometry in a large number of tissue samples from persons of differing ages. Fortunately, this need has been met by the Ekstrøm laboratory at the Radium Hospital in Oslo via the adaptation of common 96-capillary DNA sequencers to the task of parallel mutational spectrum assays 26,[73][74][75][76] .…”

“…We have tested commercial matrices like POP4 and POP6 (Applied Biosystems, Foster City, CA, USA) MegaBACE Long read matrix (GE Healthcare Bio-Sciences AB, Uppsala Sweden), GenomeLab Separation Gel LPA-1 (Beckman Coulter, Fullerton, CA, USA) and Reveal Hi Resolution Matrix (Transgenomic, Omaha, NE, USA). Additionally, in laboratory formulation of sieving matrices, by mixing or polymerization of LPA, PDMA, and PVP has demonstrated to produce suitable separation matrices used for allele separation by DCE 75 .…”