Guanosine gel for sequence‐dependent separation of polymorphic ssDNA

Abstract

The separation of fluorescent-labeled ssDNA fragments of equal length based on differences in sequence was achieved through the use of guanosine gels (G-gels) formed by guanosine-5'-monophosphate (GMP) in capillary gel electrokinetic chromatography (CGEKC) with LIF detection. Baseline resolution was achieved for homodimers and homopentamers of A, T, and C. G-gel CGEKC provided better resolution than CZE, MEKC, or a sieving gel in CGE. Resolution improved with increasing GMP, indicating that the interaction is linked to structural organization of the G-gel. Fluorescence intensity and anisotropy show that the order of interaction with G-gels is T>C>A. We then investigated four conformationally similar, polymorphic 76-mers with A/G substitutions that are utilized in forensic DNA typing. Resolution was achieved by CGEKC but not CZE or CGE. In CGEKC, the negatively charged G-gels and oligonucleotides electromigrate toward the positive inlet while being driven by EOF to the negative outlet. The net forward velocity is the greatest for oligonucleotides most closely associated with the slower, more cumbersome G-gel network. For the 76-mers, resolution increases with increasing difference in guanosine content between strands and, for a given difference, with increasing total guanosines in the strands.

Quantitative SNP genotyping by affinity capillary electrophoresis using PEG‐oligodeoxyribonucleotide block copolymers with electroosmotic flow

“…Thus far, the SNP detection methods based on ACE have employed various separating media, such as hydrogels , nanoparticles , and polymers . In 2006, Krylov and coworkers reported an ACE using a MutS protein as an affinity probe, which can bind specifically to a single‐base‐mismatched site within dsDNA .…”

“…There are two methodologies for introducing a probe ODN into a CE system. First, the probe ODN is fixed in a stationary phase or a pseudostationary phase by covalently tethering to the inner surface of a capillary tube or a sieving matrix , respectively. Consequently, the probe ODN is prohibited from migrating during electrophoresis of the sample DNA.…”

“…Such chemical modification permits the probe ODN to migrate with an attenuated velocity compared to that of an unmodified ODN. These studies have successfully demonstrated that the target ssDNA is clearly separated from its single base substituted ssDNA (nontarget ssDNA), allowing for facile SNP detection .…”

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

“…Previous work in our group demonstrated the separation of four ssDNA 76‐mers differing by only a few A/G base substitutions using guanosine‐5′‐monophosphate (GMP) in CE . The circular dichroism spectra of the four strands were essentially identical and corresponded to random strands without secondary structure, which indicated that the separation was based on sequence alone rather than conformational differences.…”

Aspergillus Collagen-Like Genes ( acl ): Identification, Sequence Polymorphism, and Assessment for PCR-Based Pathogen Detection

“…Linear gels are viscous additives that provide good separation performance and do not need to be polymerized in the capillary. Issues associated with the introduction of highly viscous materials into narrow-bore capillaries have spawned the use of thermally reversible sieving materials (76)(77)(78), including phospholipid nanogels (36). These materials are introduced into the capillary at a temperature that generates low viscosity and then converted into a highly viscous separation additive in the capillary by changing the separation temperature.…”