Incorporation of guanosine gels into sieving matrices for length‐ and sequence‐based separation of DNA in capillary electrophoresis

Abstract

Sieving gels are used in capillary gel electrophoresis to resolve DNA strands of different lengths. For complex samples, however, such as those encountered in metagenomic analysis of microbial communities or biofilms, length-based separation may mask the true genetic diversity of the community since different organisms may contribute same-length DNA with different sequences. There is a need, therefore, for DNA separations based on both the length and sequence. Previous work has demonstrated the ability of guanosine gels (G-gels) to separate four single-stranded DNA 76-mers that differ by only a few A/G base substitutions. The goal of the present work is to determine whether G-gels could be combined with commercial sieving gels in order to simultaneously separate DNA based on both length and sequence. The results are given for the four 76-mers and for a standard dsDNA ladder. Commercial sieving gels were used alone and in combination with G-gels. For the 76-mers, the combined medium was less efficient than the G-gel alone but was able to achieve partial resolution. The combined medium was at least as effective as the sieving gel alone at resolving the denatured DNA ladder and showed indications of sequence-based resolution as well, as supported by MALDI-MS. The results show that the combined sieving gel/G-gel medium retains the selectivity of the individual media, providing a promising approach to simultaneous length- and sequence-based DNA separation for metagenomic analysis of complex systems.

Sequence‐based separation of single‐strandedDNAusing nucleotides in capillary electrophoresis: Focus on phosphate

“…The results appear to rule out several possible explanations for the sequence‐based separation of the 76‐mers in this work. Contrary to our original hypothesis that the separations are due to interactions of the ssDNA with higher order G‐quadruplex structures arising from self‐assembly of GMP , this cannot be the case since AMP and UMP, which do not form such higher order structures, achieved the same results as GMP. Although the sugar in the nucleotides appears to promote the interactions that lead to separation, no separation is achieved using sugar alone.…”

“…The DNA sample was prepared in formamide and thermally denatured prior to injection into the CE. Strands 1.1–1.4 were separated in the same order as previously reported , with migration time increasing with increasing A/decreasing G in the strands. Strands 2.1–2.4, in which other nucleotides were substituted for G, were separated into three peaks.…”

“…The sequences of the ten single‐stranded 76‐mers are shown in Table . Strands 1.1–1.4 are the original four strands of human STR polymorphisms used in previous work . These tetranucleotide repeat polymorphisms are composed of TCTA/TCTG repeats and have many natural variants.…”

“…To further investigate the sequence selectivity of GMP and the basis of the DNA separation by sequence, in the present work we designed a set of ten ssDNA 76‐mers, including the four strands used in the previous work , variations of the four in which G was randomly replaced by A, T, or C to create G‐free sequences, a scrambled sequence variant of one of the original strands, and a random, unrelated sequence. We investigated the effects of treatments to denature the ssDNA sample prior to CE injection and of experimental conditions on the separation of the ten 76‐mers using GMP.…”

CGE‐laser induced fluorescence of double‐stranded DNA fragments using GelGreen dye

“…Although with lower throughput capability than the aforementioned techniques, CGE is also well suited for the simultaneous analysis of multiple DNA sequences due to its high resolving power, analysis time, and simplicity. Last developments in CGE include novel sieving matrices and additives to provide high resolution in DNA separations; novel capillary coatings ; and miniaturization into microchips . The use of LIF in CGE improves dramatically both; the LOD and linear dynamic range obtainable compared with that of UV detection .…”